Figure 4. INS-1 insulin signaling from the gut modulates AWC and AIA temperature responses to disrupt negative thermotaxis upon starvation.

(A, D, E) Mean thermotaxis bias of animals of the indicated genotypes. ins-1 was knocked out cell-specifically via Cre-Lox-mediated recombination using ins-1 alleles flanked with loxP sequences, and cell-specific expression of nCre (A) (Figure 4—figure supplement 1A–B, Supplementary file 1). Promoters used to drive Cre::SL2::GFP expression were ins-1 (ins-1-expressing cells), gcy-28d (AIA), odr-2b(3a) (AIB and AIZ), and ifb-2 (intestine). Alleles used in (D) were ins-1(nr2091), daf-18(ok480), daf-16(m26), and daf-2(e1368). DAF-16 was depleted in AWC via auxin-induced degradation of a degron-tagged daf-16 allele and AWC-specific expression of TIR1 under the ceh-36prom2_del1ASE promoter (E) (Figure 4—figure supplement 2B). Auxin was added during starvation and to the assay plate, or to the assay plate alone, as indicated in (E). Each dot represents the thermotaxis bias of a biologically independent assay comprised of 15 animals. Errors are SEM. *** and ** indicate different from fed p<0.001 and p<0.01, respectively (Student’s t-test). n.s. – not significant. P-values in red indicate t-statistic from posthoc effect size comparisons (Dunnett’s test) between the indicated genotypes (A) or the conditions (E), respectively, on the magnitude of the feeding state effect. Wild-type data in (E) were interleaved with experimental data in Figure 4—figure supplement 2C, and are repeated. (B) Representative image of the expression pattern of an ins-1p::GFP::PEST reporter in a well-fed adult hermaphrodite. Expression in the gut and in AIA is indicated by an arrow and arrowhead, respectively. Anterior is at left. Scale bar: 20 μm. (C) Schematic of starvation-dependent inhibition of the canonical insulin signaling pathway via ILP-mediated antagonism of DAF-2. (F, G) Intracellular calcium dynamics in AWC (F) and AIA (G) neurons in ins-1 mutants expressing GCaMP3 (AWC) or GCaMP5A (AIA) in response to a linear rising temperature stimulus (black lines) at 0.05 °C/s. Each row in the heatmaps displays responses from a single neuron from different animals ordered by the time of the first response; n = 19 (AWC, each fed and starved), 20 (AIA, fed) and 19 (AIA, starved). (Bottom) Each bar in the histograms represents the percentage of neurons from animals of the indicated genotypes responding during 15 s bins. Open bars indicate data from wild-type neurons repeated from Figure 2C (AWC) and Figure 3B (AIA). (H) Cumulative distribution fraction plots of total duration of calcium responses per AWC (top) and AIA (bottom) neuron calculated from data shown in F and G, respectively. Dashed lines indicate wild-type data repeated from Figure 2C (AWC) and Figure 3C (AIA). Distributions were compared using the Kolmogorov-Smirnov test. (I) Working model of AWC- and AIA-mediated disruption of negative thermotaxis upon starvation. AFD and AIY temperature responses are indifferent to feeding state. In fed animals, low and high activity in AWC and AIA, respectively is permissive for AFD-mediated negative thermotaxis. Upon prolonged starvation, INS-1 signaling from the gut acts directly or indirectly on AWC via DAF-16 to increase temperature responses. Glutamatergic signaling from AWC inhibits AIA resulting in temperature change-uncorrelated reversals and turns and disruption of AFD-mediated negative thermotaxis. Also see Figure 4—figure supplement 1 and Figure 4—figure supplement 2.

Figure 4—figure supplement 1. INS-1 insulin signaling from the intestine mediates starvation-dependent thermotaxis behavioral plasticity.

(A) Schematic of Cre-Lox-mediated recombination strategy for cell-specific knockout of ins-1. loxP sequences were inserted flanking the ins-1 genomic sequence via gene editing. nCre was expressed under cell-specific promoters (Supplementary file 1). (B) Representative images of cell- and tissue-specific reporter expression driven by Cre-mediated recombination from the heIs105 recombination reporter (Ruijtenberg and van den Heuvel, 2015). (Top) Representative images upon nCre and CFP expression under the gcy-28d and odr-2b(3a) promoters. Arrows indicate colocalization of CFP and excision-dependent GFP expression in AIA, and AIB/AIZ. Neurons were also identified by their stereotypical soma positions and axon trajectories. Scale bar: 20 μm. (Bottom) Representative images of the head alone and the body of animals expressing nCre under the ifb-2 promoter. Arrow indicates excision-dependent GFP expression in the gut; asterisks indicate mCherry expression in coelomocytes from the coinjection marker. Scale bars: 100 μm. (C) Average pixel intensity of ins-1p::GFP::PEST fluorescence in the anterior intestine of well-fed and starved (3 hr) animals. Each dot is the average fluorescence intensity in a single animal. Errors are SEM. n.s. – not significant.

Figure 4—figure supplement 2. INS-1 targets AWC to alter temperature responses as a function of satiety state.

(A, C, D) Mean thermotaxis bias of fed and starved animals of the indicated genotypes (see Supplementary file 1 for alleles used). Each dot represents the thermotaxis bias of a biologically independent assay comprised of 15 animals. Errors are SEM. DAF-16 was depleted in ASI via auxin-induced degradation of a degron-tagged daf-16 allele and ASI-specific expression of TIR1 under the srg-47 promoter (C). Data from two independent transgenic strains are combined (C). Auxin was added during starvation and to the assay plate (C). ***, ** and * indicate different from fed at p<0.001, p<0.01, and p<0.05, respectively (Student’s t-test). n.s. – not significant. Wild-type data in A and D were interleaved with experimental data in Figure 3I and Figure 3—figure supplement 1E, and are repeated. Wild-type data in C were interleaved with experimental data in Figure 4E, and are repeated. (B) Schematic of auxin-induced degradation strategy. The daf-16 gene edited allele was a kind gift of Oliver Hobert (Columbia University) (Aghayeva et al., 2020). TIR1-encoding sequences were expressed under cell-specific promoters (Supplementary file 2). (E, F) Average duration of individual response events (dots) per AWC (E) and AIA (F) neuron in fed and starved wild-type and ins-1 mutants. Wild-type data are repeated (open circles) from Figure 2—figure supplement 1D (E) and Figure 3—figure supplement 1C (F). ins-1 data are derived from Figure 4F and G. Means are indicated by horizontal black lines; errors are SEM. p-values were obtained using the Mann-Whitney U test.