Fig 1.
The genomic organization of the flp-1 gene.
flp-1 consists of six exons encoding seven FLP-1 peptides and one related non-FLP peptide; exons 3 to 6 contain the peptide-coding regions. The flp-1 allele, yn2, is a 1.1 kbp deletion that removes 567 bp of the promoter region and extends through most of exon 4. No yn2 transcripts were detected by reverse transcription-polymerase chain reaction. The ok2811 mutation disrupts the splice donor site of exon 1, leading to improper splicing and the inclusion of intronic sequence in the transcript. Only one transcript was detected. The reading frame is disrupted, suggesting that ok2811 is a null allele. The ok2781 deletion disrupts the splice acceptor site of exon 2, leading to the use of a cryptic splicing site and inclusion of intronic sequence in the transcript; only one transcript was detected. The reading frame is maintained. The deletion in ok2505 extends from intron 2 to intron 3; two transcripts are produced, both of which maintain the reading frame but result in the loss of two to four peptides relative to transcript A. flp-1 and daf-10 exons are indicated as teal and grey boxes, respectively; only four of the 18 daf-10 exons are shown. The arrows indicate the direction of transcription; the bars indicate the extent of the deletions.
Table 1.
Peptides encoded by different wild-type flp-1 transcripts and predictions for FLP-1 peptides produced in mutants.
Fig 2.
Loss of flp-1 decreases the nose touch response.
All flp-1 and daf-10 mutant alleles showed a decreased nose touch response. The decreased nose touch response in daf-10 mutants was slightly enhanced when flp-1 was also lost, as shown in the yn2 double mutants. Note that the ok2781 allele acts as a partial loss-of-function, despite the peptide coding region being intact. Mean±SEM, at least 72 animals were tested for each strain; each strain was tested in at least six independent trials; solid, colored bars (except black) represent mutant alleles, hatched bars represent transgenic lines. Ex alleles indicate a transgenic animal with an extrachromosomal array, while Is alleles indicate transgenic animals with integrated transgenes. Significantly different from wild type: ***, p≤0.001, Dunn’s post hoc test; transgenic line significantly different from mutant allele: +++, p≤0.001, ++, p≤0.01, +, p≤0.05, Dunn’s posthoc test.
Table 2.
Loss of flp-1 causes multiple defects.
Fig 3.
FLP-1 peptides modulate locomotion.
Loss of flp-1 causes hyperactivity, while overexpression of flp-1 decreases activity. Both the amplitude of the sinusoidal waveform (A) and movement rate (B) were affected. In addition, while exogenously applied serotonin inhibits swimming in wild-type animals, flp-1 mutants continued to swim in the presence of serotonin (C). daf-10 IFT-A mutants showed a decreased locomotory activity and were responsive to the inhibitory effects of serotonin. Mean±SEM, solid, colored bars (except black) represent mutant alleles, hatched bars represent transgenic lines. Ex alleles indicate a transgenic animal with an extrachromosomal array, while Is alleles indicate transgenic animals with integrated transgenes. Significantly different from wild type: ***, p≤0.001, **, p≤0.01, Tukey’s post hoc test; transgenic line significantly different from mutant allele: +++, p≤0.001, ++, p≤0.01, Tukey’s post hoc test, each strain was tested in at least three independent trials.
Fig 4.
Loss of sensory input and flp-1 signaling is responsible for the wandering behavior.
Wild-type animals remain on a bacterial food source; animals that move off a food source and do not return are considered wanderers. daf-10 flp-1 and tax-4; flp-1 double mutants are wanderers, while the single flp-1, daf-10, and tax-4 mutants do not wander. For the tax-4(p678) and tax-4(p678); ok2811 strains, animals were not scored at each time point, giving rise to some fluctuation in the data. 3–5 trials for each strain; at least 30 animals were tested for each strain.
Fig 5.
Modulation of flp-1 affects reproductive functions.
Loss and overexpression of flp-1 decreased egg-laying rates (A), suggesting that different FLP-1 peptides modulate the egg-laying circuit differently. Loss and overexpression of flp-1 generally increased the number of eggs retained in the uterus (B), again suggesting that individual FLP-1 peptides affect egg retention differently. Loss of daf-10 IFT-A function also affects reproduction. Mean±SEM, at least 43 or 24 mutants or transgenic mutants, respectively, were tested in at least 3 independent trials; solid, colored bars (except black) represent mutant alleles, hatched bars represent transgenic lines. Ex alleles indicate a transgenic animal with an extrachromosomal array, while Is alleles indicate transgenic animals with integrated transgenes. Significantly different from wild type: ***, p≤0.001, Tukey’s post hoc test; transgenic line significantly different from mutant allele: +++, p≤0.001, ++, p≤0.01, +, p≤0.05, Tukey’s post hoc test. ND = not determined.