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Figure 6. Members of a lineage-specific expansion of Ependymin-related proteins are subject to circalunar regulation on RNA and protein level. ; (a) Unrooted maximum likelihood phyolgenetic tree of Ependymin-related proteins based on the analysis published by Suárez-Castillo and García-Arrarás, 2007 including eleven of 15 Platynereis Ependymin-related proteins (ERP4.1–4.10; ERP3; blue boxes) and various additional invertebrate Ependymin sequences. The phylogeny reveals four distinct groups of Ependymin-related proteins (ERP) that are termed ERP1, ERP2, ERP3/4 and fish-specific ERPs; group-specific expansions of ERPs is observed at several places in the phylogeny. All identified Platynereis Ependymins (blue) fall into the ERP3/4 clade. A full tree with un-collapsed nodes, the alignment, NCBI accession numbers and the Platynereis sequence identifiers are provided in Figure 6—figure supplement 1 and Figure 6—source datas 1 and 2. (b,b’) Protein and RNA-Seq expression profiles validate Platynereis ERP4.9 as a new target of circalunar phase on RNA and protein level. (b) Normalised protein expression profile; (b’) DESeq2 normalised RNA expression profile. The graphs show the arithmetic mean with standard deviation, together with individual data points. (c–e) RNA expression pattern of erp4.9 in immature adult Platynereis heads. a.e.: anterior eye; p.e.: posterior eye; n.o.: nuchal organ. (c) Dorsal view, anterior to the top. Characteristic expression is observed around the dorsal blood vessel (central arrow), anterior of the nuchal organ (arrowheads) and next to the anterior eyes (arrows). Scale bar: 250 µm. (d) Dorsal view of the base of a prostomial cirrus, scale bar: 50 µm. (e) Ventral view, anterior to the top. Expression is observed in a plate-like structure at the bottom of the head, as well as around the mouth opening (arrows) and the palps (arrow heads). Scale bar: 250 µm. For additional circalunar-regulated ERPs see Figure 6—figure supplement 2 and Figure 5—source datas 16 and 17.

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Figure 1. Identification of neuronal populations required for yeast feeding. ; (A) Number of sips from 10% yeast (left) and 20 mM sucrose (right) by mated female flies of the genotype Ir76b-GAL4,UAS-GCaMP6s, fed for 3 days on a holidic diet with the indicated composition. (B) Yeast preference index (YPI) of yeast-deprived female flies in which 1261-GAL4 neurons were acutely silenced and corresponding controls. (C) Expression pattern of 1261-GAL4 in the head and tarsus of the fly. Boxes are magnifications of the antenna or the labellum. Note absence of signal in the maxillary palps. Green represents GFP signal and gray the cuticular autofluorescence. (D) YPI of yeast-deprived female flies in which Ir76b- or Ir25a-GAL4 neurons were acutely silenced and corresponding controls. (E) YPI of yeast-deprived female flies in which 1261-GAL4 neurons were all silenced, or with Ir76b-GAL80, and corresponding controls. Arrows indicate presence/loss of GFP expression in the SEZ. (D) and (E) Expression pattern of experimental flies in the brain as visualized using UAS-CD8::GFP in green, with nc82 synaptic staining in gray. Note absence of cell bodies in the Ir25a-GAL4 brain. A, antenna; VCSO, ventral cibarial sense organ; LSO, labral sense organ; L, labellum; AL, antennal lobes; SEZ, subesophageal zone. In this and following figures, empty and filled black circles represent absence and presence of the indicated elements, respectively. In (A), boxes represent median with upper/lower quartiles. In (B), (D) and (E), circles represent yeast preference in single assays, with line representing the median and whiskers the interquartile range. ***p

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Figure 2. Connections between olfactory receptor neurons and antennal lobe neurons revealed by TRACT. ; (a) Olfactory receptor neurons (ORNs) labeled in red, have their cell bodies located into two peripheral sensory organs, antennae (illustrated here) and maxillary palps, and have axons that project into the antennal lobe (AL) in the brain. All axons from ORNs expressing the same olfactory receptor converge in two glomeruli (one in each hemisphere) within the AL (red circles). There are two main types of neurons (green) in the antennal lobe, projection neurons (PNs) and local neurons (LNs). PNs have dendrites that branch in glomeruli and axons that project towards the mushroom body (MB) and/or the lateral horn (LH). (b, c) There are two types of PNs, uniglomerular PNs (uniPNs) and multiglomerular PNs (multiPNs). (b) The dendrites of uniPNs branch in a single glomerulus, and their axons project into the MB and LH via the medial antennal lobe tract (mALT). (c) The dendrites of multiPNS branch into multiple glomeruli, and their axons project into the LH via the mediolateral ALT (mlALT). (d) LNs are axonless neurons and their dendrites branch into many (or most) of the glomeruli. (e) Detection of synaptic contacts between olfactory receptor neurons and antennal lobe neurons in the adult Drosophila antennal lobe with the nlgSNTG4 receptor driven by nSybE enhancer (top panels) and GH146 enhancer (bottom panels). Induction of GFP expression in neurons (arrows) surrounding the antennal lobe when the ligand (nSyb::CD19) was driven by the orco driver in ORNs. Arrowheads indicate the axons of PNs in mALT and mlALT. Left: nSyb::CD19+ axons from ORNs (red); middle: GFP+ neurons in the antennal lobe (green); right: merged images of nSyb::CD19 and GFP. In the brains with the nlgSNTG4 receptor driven by the nSybE enhancer (top panels) GFP expression was induced in different neuronal types, including uniPNs, LNs and and several multiPNs at the ventral part. In the brain with the nlgSNTG4 receptor driven by the PN-specific driver GH146 (bottom panels), only PNs were GFP+. Most of these cells are uniPNs, but there was also one GFP+ multiPN in each antennal lobe (yellow arrow). Maximum projection of z-stack confocal images. Scale bar = 50 µm. See Supplementary file 2 for additional information.

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Figure 1—figure supplement 2. Characterization of neuronal populations required for yeast feeding. ; (A) YPI of w1118 female flies prefed a complete diet (YBM) or deprived of yeast for 3 days. (B) Overview of the two-color food choice assay. (C) Experimental design for the neuronal silencing experiments using shibirets. Flies were reared and yeast-deprived at 18°C until two hours prior to the assay. Experimental flies were tested at 18°C and 30°C. Genetic control flies were tested at 30°C. (D) and (E) Expression pattern of Ir76b- (D) and Ir25a-GAL4 (E) in the head and tarsus of the fly. Boxes are magnifications of the antenna or the labellum. Note absence of signal in the maxillary palps. Green represents GFP signal and gray the cuticular autofluorescence. (F) Expression pattern of 1261-, Ir76b- and Ir25a-GAL4 lines in the ventral nerve cord (VNC). Green represents GFP signal and gray nc82 synaptic staining. Note absence of cell bodies in the Ir25a-GAL4 VNC. (G) Number of sips from 10% yeast (upper) and 20 mM sucrose (lower) by yeast-deprived females in which 1261-, Ir76b- or Ir25a-GAL4 neurons were acutely silenced, and corresponding controls. (H) Number of sips from 10% yeast by amino acid-deprived females (left) and from 20 mM sucrose by sucrose-deprived females (right) in which 1261-GAL4 neurons were acutely silenced, and corresponding controls. (I) YPI of yeast-deprived female flies in which Ir25a-GAL4 neurons were all silenced, or with Ir76b-GAL80, and corresponding controls. Expression pattern of experimental flies in the brain as visualized using UAS-CD8::GFP in green, with nc82 synaptic staining in gray. Arrows indicate presence/loss of GFP expression in the SEZ. FL, foreleg projections; ML, midleg projections; HL, hindleg projections; W, wing projections. In (A) and (I), circles represent yeast preference in single assays, with line representing the median and whiskers the interquartile range. In (G) and (H), boxes represent median with upper/lower quartiles. ***p

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