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. Author manuscript; available in PMC: 2014 Dec 8.
Published in final edited form as: Science. 2014 Mar 28;343(6178):1516–1520. doi: 10.1126/science.1251285

Fig. 4. Physiological connectivity does not ensure molecular clock coupling in the lateral neuron network.

Fig. 4

(A to C) Immunostaining of PER protein in PDF positive and negative lateral neurons across different time-points on day 4 of constant darkness (DD4). In UAS-DBTL control fly brains, PER accumulated in the PDF positive (s-LNvs) and negative (LNds and 5th s-LNv) neurons with the same phase (A). In Pdf>DBTL flies in which the PDF neurons were slowed down, only two LNds (marked by yellow arrows) were coupled with the s-LNvs, displaying a shifted phase of PER cycling relative to the other LNds (B). In Pdfr-, Pdf>DBTL flies, all PDF negative neurons (LNds and 5th s-LNv) had similar phases of PER cycling, and none were coupled to the uniformly delayed s-LNvs (C). Scale bars, 5 μm. (D to F) Quantification of PER immunostaining intensity within lateral neurons of UAS-DBTL flies (D), Pdf>DBTL flies (E), and Pdfr-, Pdf>DBTL flies (F). The LNds in (E) were divided into two groups based on their phase differences and quantified separately: the two LNds coupled to the s-LNvs were quantified as “LNd (shifted)” and the others as “LNd (unshifted)”. (G to J) The two shifted LNds express neuropeptide sNPF. LNds were co-immunostained for PER and sNPF at CT0 and CT12 on DD4 (CT0 and CT12 correspond to the light-on and light-off times had the 12hr: 12hr light: dark cycles continued). In UAS-DBTL control flies, the two sNPF+ LNds and four sNPF- LNds had similar subcellular PER distribution (G) and expression levels (I) at each of these time-points. In Pdf>DBTL flies, the sNPF+ LNds differ in their PER distribution (H, yellow arrows) and intensity (J) from the sNPF- LNds. Scale bars, 5 μm. Asterisks in (G) and (H) indicate sNPF+ cells that are not clock neurons (lack of PER expression). The letters ‘a’ and ‘b’ in (I) and (J) denote significantly different groups (p < 0.0001 for both), by Kruskal-Wallis one-way ANOVA and Dunn’s multiple comparisons test. Sample sizes are reported in Table S6 for (D to F), and in Table S7 for (I and J).