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. 2016 Mar 7;5:e12499. doi: 10.7554/eLife.12499

Figure 2. The AP2 transcription factor APTF-1 controls FLP-11 expression in RIS.

(A) Transcriptional analysis of aptf-1(gk794) mutants revealed genes that are regulated by APTF-1. Wild-type and aptf-1(gk794) pretzel-stage embryos and sleeping L4 larvae were used for a transcriptome analysis. In both life stages, expression of the FMRFamide-like neuropeptide FLP-11 was strongly reduced in aptf-1(gk794). This suggests transcriptional control of FLP-11 by APTF-1. Data can be found in Supplementary file 1, Tables 1A and 1B. (B) Expression of pflp-11::mKate2 in wild type and aptf-1(gk794). Expression of mKate2 was absent in RIS in aptf-1(gk794) showing that APTF-1 controls expression of FLP-11. Expression of flp-11 in RIS was reminiscent to the expression of the flp-11 homolog afp-6 in RIS in Ascaris nematodes (Yew et al., 2007). Expression for additional genes can be found in Figure 2—figure supplements 2 and 3. (C) flp-11 expression profile in RIS over the sleep-wake cycle. Expression does not change with the sleep-wake cycle. (D) Probability distribution of nose speeds during wake and sleep for wild type, aptf-1(gk794) and aptf-1(gk794); paptf-1::tfap2β rescue. (E) Comparison of immobility during sleep for wild type, aptf-1(gk794), and aptf-1(gk794); paptf-1::tfap2β. The mouse TFAP2β partially rescued the aptf-1(gk794) sleep phenotype. (F) Comparison of pflp-11::GFP fluorescence intensity in RIS for wild type, aptf-1(gk794), and aptf-1(gk794); paptf-1::tfap2β. The mouse TFAP2β partially rescued the expression of flp-11 in RIS (18% of wild-type level). (G) Analysis of putative AP2-binding sites in the flp-11 promoter region. The flp-11 promoter region was scanned for the primary mouse AP2α-binding site. Overlap was found (p<0.001, q=0.06 (Grant et al., 2011)) for one binding site. Statistical test used was Wilcoxon Signed Paired Ranks test. ** denotes statistical significance with p<0.01, *** denotes statistical significance with p<0.001. Scale bar is 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.12499.005

Figure 2.

Figure 2—figure supplement 1. C10C6.7 is a putative four transmembrane helix protein that is expressed in RIS and that is controlled by aptf-1.

Figure 2—figure supplement 1.

(A) Bioinformatics analysis suggests that C10C6.7 is a four transmembrane helix protein (Krogh et al., 2001). (B) Expression pattern of GFP-tagged fosmids for C10C6.7. Expression is visible in nine cells: interneuron RIS; pharyngeal neurons M1, M2, Motor-interneuron (M-IN), an unidentified pair of pharyngeal neurons (**) and an unidentified pair of sensory neurons (*). Expression of C10C6.7 protein in RIS is controlled by aptf-1. (C) Probability distribution of nose speeds during wake and sleep for C10C6.7(goe3) and C10C6.7(goe5) shows that C10C6.7 does not play a significant role in sleep control. Statistical test used was Wilcoxon Signed Paired Ranks test. Scale bars are 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.12499.006
Figure 2—figure supplement 2. Expression pattern of sto-3 and H19N07.3.

Figure 2—figure supplement 2.

(A) Expression pattern of sto-3 promoter fusions. STO-3 is expressed in RIB neuron and additionally in three unidentified non-neuronal cells in the tale (not shown). (B) Probability distribution of nose speeds during wake and sleep for sto-3(tm1488) shows that sto-3 does not play a significant role in sleep control. (C) Expression pattern of GFP-tagged fosmids for H19N07.3. The H19N07.3 protein is expressed in all somatic cell nuclei, but its levels are not regulated by aptf-1. Statistical test used was Wilcoxon Signed Paired Ranks test. Scale bars are 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.12499.007
Figure 2—figure supplement 3. FLP-11 is strongly expressed in RIS and weakly in additional neurons. APTF-1 controls the expression in RIS.

Figure 2—figure supplement 3.

(A) Expression pattern of ynIs40(pflp-11::GFP) in wild type and aptf-1(gk794) mutant. The transgene expresses in several neurons including RIS. aptf-1(gk794) abolishes the expression specifically in RIS. (B) Expression pattern of goeIs288(pflp-11::mKate2) in wild type and the aptf-1(gk794) mutant. Strong expression is visible only in RIS. By increasing the contrast to the point where RIS is over-saturated several additional neurons becomes visible that may be identical to those seen in ynIs40 (Kim and Li, 2004). aptf-1(gk794) strongly reduces the expression specifically in RIS. Scale bars are 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.12499.008
Figure 2—figure supplement 4. Mouse TFAP2beta partially restores expression of flp-11 neuropeptides in RIS in aptf-1 mutant worms.

Figure 2—figure supplement 4.

Expression of pflp-11::GFP in wild type, aptf-1(gk794), and aptf-1(gk794); tfap2beta rescue. In aptf-1(gk794), expression of flp-11 is strongly reduced but could partially be restored by the mouse TFAP2beta. Expression of flp-11::GFP in the rescue strain varied between 5–50% of wild-type levels. Here, we show a picture of 50% rescue. Scale bar is 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.12499.009