Regulation of Caenorhabditis elegans male mate searching behavior by the nuclear receptor DAF-12

Abstract

Coordination of animal behavior with reproductive status is often achieved through elaboration of hormones by the gonad. In the nematode Caenorhabditis elegans, adult males explore their environment to locate mates. Mate searching is regulated by presence of mates, nutritional status, and a signal from the gonad. Here we show that the gonadal signal acts via the nuclear receptor DAF-12, a protein known to regulate several C. elegans life-history traits. DAF-12 has both activational and organizational functions to stimulate exploratory behavior and acts downstream of the gonadal signal, outside of the gonad. DAF-12 acts upstream of sensory input from mating partners and physiological signals indicating nutritional status. Mate searching was rescued in germ-line ablated animals, but not if both germ line and somatic gonad were ablated, by a precursor of the DAF-12 ligand, dafachronic acid (DA). The results are interpreted to suggest that the germ line produces a DA precursor that is converted to DA outside of the germ line, possibly in the somatic gonad. As it does in other pathways in which it functions, in regulation of male mate searching behavior DAF-12 acts at a choice point between alternatives favoring reproduction (mate searching) vs. survival (remaining on food).

Figure 1.—

The DAF-12 pathway regulates male exploratory behavior. The rate of male exploratory behavior is determined by the leaving assay. For various genotypes and treatments, the fraction of (usually 20) males that have not yet traveled 3 cm away from a 1-cm patch of E. coli food (nonleavers) is scored at various times after the start of the assay. The points on the graphs show the individual observations. The leaving rate, taken as a measure of the tendency for exploratory behavior, is calculated as the probability of leaving per hour, P_L. The data are analyzed using the R survival package (http://cran.r-project.org/web/packages/survival/index.html) to fit the censored data to an exponential parametric survival model, using maximum likelihood. The slopes of the straight lines through the data illustrate the constant hazard rate (λ), taken here as P_L. The results are compiled in Table 1 and summarized in Figure 2 for the various daf-12 alleles. (A) daf-12 gene function is required for a wild-type rate of exploratory behavior. Males homozygous for a daf-12 strong loss-of-function mutation leave food more slowly than wild-type males. (B) The DAF-12 ligand DA stimulates exploratory behavior. Males homozygous for daf-9(m540), a hypomorphic allele of the daf-9 biosynthetic gene required for synthesis of DA, leave food more slowly than wild-type males. Leaving rate is restored by treatment of daf-9 animals with DA. (C) Evidence a DAF-12/DIN-1 corepressor complex inhibits leaving. The structures of the daf-12 alleles shown are illustrated in Figure 2. Successive deletion of the DAF-12 C-terminal LBD results in decreased leaving rate. Introduction of a point mutation that prevents DIN-1 binding [daf-12(rh61dh115)] restores wild-type leaving rate. Wild-type data are the same as in A. (D) The DAF-12 N-terminal DBD stimulates exploratory behavior. The structure of the daf-12 alleles shown is illustrated in Figure 2. Alleles of daf-12 with deletions covering the entire LBD including the DIN-1 binding region have a high rate of leaving. Wild-type data are the same as in A. (E) daf-12 has an adult function to stimulate exploratory behavior. Males homozygous for a temperature-sensitive allele of daf-12 have reduced leaving rate when shifted to nonpermissive temperature as adults. Quantitative analysis of the data in A–D is given in Table 1. Data for the series of daf-12 alleles shown in A, C, and D were compared to pooled data for wild type.

Figure 2.—

Correspondence of DAF-12 protein structure with exploratory behavior for a series of C-terminal deletion alleles. Deletion of the LBD excluding the corepressor binding region decreases exploratory behavior. Introduction of a point mutation that abolishes DIN-1 binding (red “x”) restores exploratory behavior, as does deletion of the LBD together with the DIN-1 binding region. Error bars are 95% confidence intervals.

Figure 3.—

Relationship between stimulation of exploratory behavior by daf-12 and that of other signaling pathways that regulate male exploratory behavior. (A) Hermaphrodites inhibit exploratory behavior of males homozygous for ligand-independent daf-12(m20). The effect of five paralyzed (unc-51) hermaphrodites on mate searching is shown. (B) A signal indicating nutritional status inhibits exploratory behavior of males homozygous for ligand-independent daf-12(m20). The effect of 12-hr food deprivation on wild-type vs. daf-12(m20) males is shown. Data are given for the first 8 hr of the leaving assay and censored thereafter since after this time the leaving rate returns to the fed rate. (C) The gonad requires daf-12 function to stimulate exploratory behavior. In males homozygous for the daf-12 presumptive null allele m583, ablation of the gonad has little or no effect on leaving rate. (D) Ligand-independent daf-12(m20) stimulates exploratory behavior independently of the gonad. Wild-type mock ablated and gonad ablated data are the same in C and D.

Figure 4.—

Stimulation of male exploratory behavior by the DA precursor after germ-line but not whole gonad ablation. (A) The DA precursor can rescue germ-line ablated adult males. (B) The DA precursor can rescue germ-line defective mes-1(bn7) males. (C) In gonad ablated males the DA precursor cannot rescue the mate searching defect. Wild-type mock ablated data are the same as in A. (D) The DA precursor fails to rescue mate searching in the daf-12 null animals. (E) The DA precursor fails to rescue daf-9(m540) adult males. Wild-type data are the same as in D.