Figure 1.
Commitment to dauer or reproductive development.
(A) Developmental molt times of C. elegans N2 strain growing in favorable (blue) or unfavorable (red) conditions. (B–E) Time courses of commitment as a function of environmental conditions (pheromone). Top, representative colored bars indicate shifts to unfavorable conditions (red) or favorable conditions (blue). Bottom, means of dauer frequencies between biological replicates ± standard deviation. Numbers in parentheses indicate total worms per time point. (B) Period of pheromone sensitivity during L1 and L2: worms respond to pheromone between 12 and 18 hph. (C) Point of commitment to dauer: worms commit to dauer 33 hph denoted by the red dashed gridline. (D) Point of commitment from L2d to L3: worms commit to L3 after a 3 h pulse in favorable conditions when shifted at 24 hph. (E) Start time of pulse shifts to favorable conditions during L2d. Pheromone was added to worms 3 h post-shift to favorable conditions. Cultures shifted to favorable conditions at 33 hph show a higher ratio of dauers since worms commit to dauer at 33 hph (red dashed gridline). Control, worms grown without pheromone. Dauer, worms grown in 3% (v/v) pheromone with no shifts.
Figure 2.
High amounts of DA are required for normal adult development.
(A) Images of dauer, arrested L3, abnormal development Mig and Cut worms, and normal adults. Yellow hatched area encloses the gonad, v, vulva. (B) Distribution of developmental stages as a function of DA, scored 48 hph. Means of dauer (red), abnormal development (arrested L3, Mig and Cut; yellow) and normal adult (L3, L4, and young adult; blue) phenotype in daf-9(dh6) worms. (C) Distribution of developmental stages in the daf-9(rh50) background. (D) Distribution of stages in the adult fraction of phenotypes. Means of population proportions of stages indicate the relative developmental rate at each concentration of DA scored at 48 hph. Error bars represent means ± standard deviations across three biological replicates, N>500. Mig, gonad migratory defective; Cut, cuticle defective; YA; young adult. * Worms were gravid the next day.
Figure 3.
Dauer pheromone regulates the threshold for reproductive development.
(A–C) Distribution of the phenotypes of the strain daf-9(dh6) when supplemented with DA: dauers (A), abnormal adults (B), and normal adults (C), as a function of DA and pheromone. Each pixel on the heat map is the mean fraction of population (see bar on the right for quantification) developing in the specific category, N>300 per pixel. Partition of abnormal adults and adults into sub-categories is detailed in Figure S3. (D) Concentrations of DA required to surpass the dauer bypass DA threshold as a function of pheromone. Yellow dashed line indicates 90% non-dauers in the population. (E) Concentrations of DA required for normal adult development without any arrested L3, Mig, or Cut phenotypes. Blue dashed line indicates 90% normal adults in the population. (F) Regression analysis of panels D and E; points correspond to 90% non-dauer (Yellow, 3D) and 90% normal adult (Blue, 3E, see Experimental Procedures for details of regression). Normal adult development requires an additional 30 nM DA above the amount for dauer bypass. Mig, gonad migratory defective; YA, young adult.
Figure 4.
Dauer, abnormal development and normal adult fates as a function of exposure times to 100 nM Δ7-DA. (A) daf-9(dh6) worms start responding to Δ7-DA at 15 hph and require an additional 12 h of Δ7-DA for normal adult development. Top, representative colored bars indicating the shift experiment: red bars indicate EtOH carrier and blue bars indicate Δ7-DA. Bottom, normal adult (blue), abnormal adult (yellow), and dauer (red) bars indicate the population fraction per time point. (B) Worms become refractory to Δ7-DA at 33 hph, the same time that they commit to dauer. N2 indicates worms shifted from unfavorable to favorable conditions as indicated in Figure 1C, and points indicate dauer proportions (abnormal development is considered non-dauer in this panel). (C) Pulses of Δ7-DA indicate the minimal time necessary for normal development when added at 15 hph. Top, length of pulses. Bottom, normal adult (blue), abnormal development (yellow), and dauer (red) bars indicate the population fraction per time point. (D) Worms have no memory of previous exposure to Δ7-DA before the L1/L2 molt. Pie charts indicate proportions of dauers (red), abnormal development (yellow), and normal adults (blue) as a function of total amount of time exposed to Δ7-DA (x-axis) when exposed to Δ7-DA at different hours post-hatch (y-axis). N>100 for all time points.
Figure 5.
daf-9 is transcriptionally upregulated in the hypodermis after commitment to reproductive development.
(A,E) Fold change of total daf-9 transcripts quantified by qPCR during development of synchronized broods (see Experimental Procedures, Table S2). Bars represent means ± standard deviations of fold change between biological replicates. (A) Development in favorable conditions. (E) Development in unfavorable conditions. (B,F) Fraction of worms expressing hypodermal daf-9 during development in (B) favorable and (F) unfavorable conditions. (C,D) Δ7-DA regulates hypodermal daf-9 transcription and development. daf-9(dh6) animals carrying the a daf-9 promoter construct fused to gfp (pdaf-9::gfp) were grown on 0 to 100 nM DA. (C) Hypodermal GFP intensity. Animals grown in the presence of 0 to 0.5 DA show no hypodermal daf-9 expression. Higher DA levels (0.75 to 10 nM DA) result in strong hypodermal expression, whereas higher levels (50 nM) abolish expression. Each bar represents the pixel intensity of a fixed area of the hypodermis of a single animal. (D) Phenotypic distribution of daf-9(dh6) worms expressing hypodermal daf-9 when exposed to different concentrations of DA. Hypodermal upregulation is visible only at concentrations of DA that give rise to abnormal adults. (G,I) Hypodermal expression in worms grown in pheromone (red) and shifted to favorable conditions for a specified time window (blue) and shifted back to unfavorable conditions: (G) 6 h with a dauer frequency of 7%±5%, and (I) 1 h with a dauer frequency of 90%±10%. Bars represent the average of three biological replicates ± standard deviation. (H,J) Fold change of total daf-9 transcripts quantified by qPCR when worms are shifted from unfavorable to favorable conditions (H), or maintained in unfavorable conditions (J).
Figure 6.
Hypodermal daf-9 expression propagates from anterior to posterior upon commitment to the L3 fate.
(A, top) Fluorescent images of worms at each time point are shown at shift from unfavorable to favorable at 0 (24 hph) h (leftmost image) through 12 hph (rightmost image). Arrowheads mark the XXX cells. (A, bottom) Expression of hypodermal daf-9 was quantified along the anterior posterior axis in 4–6 worms in each time point. Each green shaded histogram represents the mean grey value of DAF-9::GFP per worm, normalized to length. Different worms were imaged at each time point (see Text S1 for details of analysis). (B–D) XXX cells and Δ7-DA are required to initiate hypodermal daf-9 expression and reproductive development. (B) Cells were ablated during L2d and recovered in favorable conditions. (C) L2d ablated worms were let to recover on 1, 5, or 10 nM Δ7-DA. All worms expressing hypodermal daf-9 developed into normal adults with no Mig or Cut phenotypes. (D) Worms were grown to L2d, XXX cells were ablated after commitment to L3 at 27 hph. * p<1×10−4, ** p<1×10−10.
Figure 7.
A feedback loop amplifies a DA signal leading to coordinate development.
(A) Environmental conditions overlap with DA time of action. Top, growth in favorable conditions (blue) during the induction period commits worms to adulthood. Committed worms develop into adults even if shifted to unfavorable conditions (red). Commitment to adulthood correlates with the time of action of DA. Middle and bottom: worms grown in unfavorable conditions during the induction period develop into L2d worms which decide between regular development and alternative development during the integration period (between 15 and 33 hph, hashed grey lines). Worms commit to the dauer fate at 33 hph or to adult development if exposed to favorable conditions for 3 h. Development to dauer correlates with no DA production, and development to adulthood correlates with production of DA. (B) Noisy and uncertain environmental information is measured by sensory neurons and reduced in complexity into the four signaling pathways. Information complexity is reduced further to the XXX cells, the primary source of DA. If nascent amounts of DA produced by the XXX cells bypass the dauer DA threshold, worms will develop into reproductive adults, and if DA levels are under the threshold, worms will develop into dauers. Upon commitment to reproductive development, DA originating from the XXX cells will initiate the hypodermal daf-9 positive feedback loop, thus increasing the amounts of DA and thus locking the adult decision and producing sufficient amounts of DA for complete adult development. The positive feedback loop canalizes development guaranteeing that sufficient amounts of DA are produced so that abnormal phenotypes are not expressed in adult worms.