TaDa-seq identifies novel Alk-regulated genes in the Drosophila larval CNS.

a. Schematic overview of experimental conditions comparing wild-type Alk (Ctrl) with Alk dominant-negative (AlkDN) conditions. The Drosophila Alk RTK is comprised of extracellular, transmembrane and intracellular kinase (red) domains. Upon Jelly belly (Jeb, blue dots) ligand stimulation the Alk kinase domain is auto-phosphorylated (yellow circles) and downstream signaling is initiated. In AlkDN experimental conditions, Alk signaling is inhibited due to overexpression of the Alk extracellular domain. b. The TaDa system (expressing Dam::RNA Pol II) leads to the methylation of GATC sites in the genome, allowing transcriptional profiling based on RNA Pol II occupancy. c. Pie chart indicating the distribution of TaDa peaks on various genomic features such as promoters, 5’ UTRs, 3’ UTRs, exons and introns. d. Volcano plot of TaDa-positive loci enriched in AlkDN experimental conditions compared to control loci exhibiting Log2FC<-2, p≥0.05 are shown in blue. Alk-associated genes such as mamo, C3G, Kirre, RhoGAP15B and mib2 are highlighted in purple. e. Venn diagram indicating Alk-dependant TaDa downregulated genes from the current study compared with previously identified Alk-dependant TaDa loci in the embryonic VM (Mendoza-Garcia et al., 2021). f. Enrichment of Gene Ontology (GO) terms associated with significantly down-regulated genes in AlkDN experimental conditions.

Integration of TaDa data with scRNA-seq identifies an enrichment of Alk-regulated genes in neuroendocrine cells.

a. UMAP feature plot indicating Alk (in red) and Jeb (in green) mRNA expression in a control (w1118) whole third instar larval CNS scRNA-seq dataset (Pfeifer et al., 2022). b. UMAP visualizing AUCell enrichment analysis of the top 500 TaDa downregulated genes in the third instar larval CNS scRNA-seq dataset. Cells exhibiting an enrichment (threshold >0.196) are depicted in red. One highly enriched cell cluster is highlighted (red circle). c. Heatmap representing expression of the top 500 genes downregulated in TaDa AlkDN samples across larval CNS scRNA-seq clusters identifies enrichment in neuroendocrine cells. d. UMAP indicating third instar larval CNS annotated clusters (Pfeifer et al., 2022), including the annotated neuroendocrine cell cluster (in orange). e. Matrix plot displaying expression of canonical neuroendocrine cell markers. f. Feature plot visualizing mRNA expression of Dh44, Dh31, sNPF and AstA neuropeptides across the scRNA population. g. Alk staining in Dimm-Gal4>UAS-GFPcaax third instar larval CNS confirms Alk expression in Dimm-positive cells. Alk (in magenta) and GFP (in green), close-ups indicated by boxed regions and arrows indicating overlapping cells in the central brain and ventral nerve cord. Scale bars: 100 μm.

TaDa and RNA-seq identifies CG4577 as a novel Alk-regulated neuropeptide.

a. Flowchart representation of the multi-omics approach employed in the study and the context dependent filter used to integrate TaDa, bulk RNA-seq and scRNA-seq datasets. b. Venn diagram comparing bulk RNA-seq (Log2FC>1.5, p<0.05) and TaDa datasets (Log2FC<-2, p<0.05). A single candidate (CG4577/Spar) is identified as responsive to Alk signaling. c. TaDa Pol II occupancy of CG4577/Spar shows decreased occupancy in AlkDN experimental conditions compared to control. d. Expression of CG4577/Spar in w1118 (control), AlkΔRA (Alk loss-of-function allele) and AlkY1355S (Alk gain-of-function allele) larval CNS. Boxplot with normalized counts, **p<0.01, ***p<0.001. e. Feature plot showing mRNA expression of CG4577/Spar and Alk in third instar larval CNS scRNA-seq data. Neuroendocrine cluster is highlighted (red circle). f. CG4577/Spar-PA amino acid sequence indicating the signal peptide (amino acids 1-26, in red), glutamine repeats (in green) and the anti-CG4577/Spar antibody epitopes (amino acids 211-225 and 430-445, underlined). Center lines in boxplots indicate medians; box limits indicate the 25th and 75th percentiles; crosses represent sample means; whiskers extend to the maximum or minimum.

Spar expression in the Drosophila larval brain.

a. Immunostaining of w1118 third instar larval brains with Spar (green) and Alk (magenta) revealing overlapping expression in central brain and ventral nerve cord. a’-a’’. Close-up of Spar expression (green) in ventral nerve cord (a’) and central brain (a’’). b. Immunostaining of w1118 third instar larval CNS together with the body wall muscles, showing Spar (green) expression in neuronal processes (white arrowheads) which emerge from the ventral nerve cord and innervate larval body wall muscle number 8. c-c’. Decreased expression of Spar in third instar larval brains expressing spar RNAi (C155-Gal4>Spar RNAi) compared to control (C155-Gal4>UAS-GFPcaax) confirms Spar antibody specificity (Spar in green). d-d’. Spar overexpression (C155-Gal4>UAS-Spar) showing increased Spar expression (in green) compared to control (C155-Gal4>+) larval CNS. e-e’’. Immunostaining of Dimm-Gal4>UAS-GFPcaax third instar larval brains with Spar (in magenta), GFP and Dimm (in blue) confirms Spar expression in Dimm-positive neuroendocrine cells (white arrowheads). f-i. Spar protein expression in w1118, AlkY1355S, and AlkΔRA third instar larval brains. Quantification of Spar levels (corrected total cell fluorescence, CTCF) in i. j-m. Overexpression of Jeb in the third instar CNS (C155-Gal4>UAS-Jeb) leads to increased Spar protein expression compared to controls (C155-Gal4>UAS-GFPcaax). Quantification of Spar levels (corrected total cell fluorescence, CTCF) in m. (**p<0.01; ***p<0.001) Scale bars: 100 μm. Center lines in boxplots indicate medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the maximum or minimum.

Identification of Spar peptides in Drosophila CNS tissues.

Peptides derived from the Spar prepropeptide identified by mass spectrometry in wild-type-like control flies (FM7h;hs-svr, upper panel) and svr mutant (svrPG33;hs-svr, lower panel) flies. The predicted amino acid sequence of the CG4577-PA Spar isoform is depicted for each genetic experimental background. Peptides identified by database searching (UniProt Drosophila melanogaster, 1% FDR) are marked by blue bars below the sequence. In addition, peptides correctly identified by de novo sequencing are marked by orange bars above the sequence. Red bars indicate basic prohormone convertase cleavage sites, green bar indicates the signal peptide.

Spar expression in larval neuropeptide expressing neuronal populations.

a. Immunostaining of w1118 third instar larval CNS with Spar (in magenta) and PDF (in green). Closeups (b-b’’) showing PDF- and Spar-positive neurons in central brain indicated by white arrowheads. c. Immunostaining of w1118 third instar larval CNS with Spar (in magenta) and Dh44 (in green). Closeups (d-d’’) showing Dh44- and Spar-positive neurons in central brain indicated by white arrowheads. e. Immunostaining of w1118 third instar larval CNS with Spar (in magenta) and Ilp2 (in green). Closeups (f-f’’) showing Ilp2- and Spar-positive neurons in central brain indicated by white arrowheads. g. Immunostaining of w1118 third instar larval CNS with Spar (in magenta) and AstA (in green). Closeups (h-h’’) showing AstA- and Spar-positive neurons in central brain indicated by white arrowheads. i. Immunostaining of w1118 third instar larval CNS with Spar (in magenta) and Lk (in green). Closeups (j-j’’) showing Lk (LHLK neurons)- and Spar-positive neurons in central brain indicated by white arrowheads. k. Immunostaining of w1118 third instar larval CNS together with the body wall muscles, showing Spar (in magenta) expressing Lk (in green) (ABLK neurons) in neuronal processes, which emerge from the ventral nerve cord and innervate the larval body wall muscle. Closeups (l-l’’) showing co-expression of Lk and Spar in neurons which attach to the body wall number 8 indicated by white arrow heads. Scale bars: 100 μm.

Generation of SparΔExon1 mutant and expression of Spar in circadian neurons.

a. Schematic overview of the Spar gene locus and the SparΔExon1 mutant. Black dotted lines indicate the deleted region, which includes the transcriptional start and exon 1. b. Immunoblotting for Spar. Spar protein (35 kDa) is present in larval CNS lysates from wild-type (w1118) controls but absent in SparΔExon1 mutants. c-d’. Immunostaining confirms loss of Spar protein expression in the SparΔExon1 mutant. Third instar larval (c-c’) and adult (d-d’) CNS stained for Spar (in magneta). Spar signal is undetectable in SparΔExon1. e. Expression of Spar in LNv, LNd and DN1 circadian neuronal populations, employing publicly available RNA-seq data (Abruzzi et al., 2017). f. Feature plot of Spar expression in circadian neurons, employing publicly available scRNA-seq data (Ma et al., 2021). g. Violin plot indicating Spar expression throughout the LD cycle, showing light phase (ZT02, ZT06 and ZT10) and dark phase (ZT14, ZT18 and ZT22) expression. h. Dotplot comparing Spar expression throughout the LD cycle with the previously characterized circadian-associated neuropeptide pigment dispersion factor (Pdf) and the core clock gene Period (per). Expression levels and percentage of expressing cells are indicated. i-j. Spar expression in clock neurons (Clk856-Gal4>UAS-GFPcaax) of the larval CNS (i-j), visualized by immunostaining for Spar (magenta), Alk (in blue) and clock neurons (GFP, in green). j’-j’’. Close up of central brain regions (yellow dashed box in j) indicating expression of Spar in Clk856-positive neurons (white arrowheads). k-l. Immunostaining of Clk856-Gal4>UAS-GFPcaax in adult CNS with GFP (in green), Spar (in magenta) and Alk (in blue). l’-l’’. Close ups of CNS regions (yellow dashed box in l) stained with GFP (in green) and Spar (in red) showing a subset of clock-positive neurons expressing Spar (white arrowheads). Scale bars: 100 μm.

Lifespan and activity plots of SparΔExon1 mutants.

a. Kaplan-Meier survival curve comparing AlkΔRA (n=31) and SparΔExon1 (n=30) flies to w1118 controls (n=27). Outliers from each group were determined by Tukey’s test, and statistic al significance was analyzed by Log-rank Mantel-Cox test (****p<0.0001). b. Representative activity profile graph illustrating average activity count measured every 5 min across a 24 h span. Black arrows indicate morning and evening activity peaks. Empty arrows indicate anticipatory increase in locomotor activity of SparΔExon1 mutant flies occurring before light transition. One-way ANOVA followed by Tukey’s multiple comparisons post-hoc test was used to determine significance between groups (****p<0.0001). w1118 (n=27), SparΔExon1 (n=30), AlkΔRA (n=31). c. Representative sleep profile, demonstrating the proportion of flies engaged in sleep measured at 5-minute intervals over a 24-hour period. One-way ANOVA followed by Tukey’s multiple comparisons post-hoc test was used to determine significance between groups (****p<0.0001; *p<0.05). w1118 (n=27), SparΔExon1 (n=30), AlkΔRA (n=31). d. Representative average actogram of individual flies in each group. Each row corresponds to one day, visualized as 288 bars each representing one 5 min interval. Yellow bar represents the time of the day when the lights are turned on, with ZT0 indicating the morning peak and ZT12 the evening peak. e. Mean locomotor activity per day over 30 days. One-way ANOVA followed by Tukey’s multiple comparisons post-hoc test was used to determine significance between groups (***p<0.001). w1118 (n=27), SparΔExon1 (n=30), AlkΔRA (n=31). f. Mean locomotor activity for 6 h intervals over 14 days. a.m. anticipation and p.m. anticipation depict mean activity in the 6 h before lights on and 6 h before lights off respectively. Unpaired student t-test was used to determine the significance between control and SparΔExon1 (****p<0.0001). w1118 (n=27), SparΔExon1 (n=30). g. Mean locomotor activity for 6 h intervals over 5 days in LD and 5 days in DD conditions. The a.m. and p.m. anticipation depict mean activity in the 6 h before lights on (or subjective lights on) and 6 h before lights off (or subjective lights off) respectively. Unpaired student t-test was used to determine the significance between SparΔExon1 and controls (****p<0.0001); paired student t-test was used to determine significance in each group between the two experimental conditions (****p<0.0001; ***p<0.001). w1118 (n=32), SparΔExon1 (n=31). h-h’. Mean sleep per day across a 3-day average (Day 5-7 (h), Day 20-22 (h’)). One-way ANOVA followed by Tukey’s multiple comparisons post-hoc test was used to determine significance between groups (****p<0.0001). w1118 (n=27), SparΔExon1 (n=30), AlkΔRA (n=31). The error bars in the bar graphs represents standard deviation.

SparΔExon1 mutants exhibit circadian activity disturbances

a. Representative activity profile for w1118 controls, illustrating the average activity count measured every 5 min across a 24 h span for Light-Dark (LD) for 5 cycles (black line), subsequently switching to Dark-Dark (DD) for 5 cycles (gray lines). ZT0 and ZT12 represent the start and end of the photoperiod respectively. CT0 and CT12 represent the start and end of the subjective day in constant dark conditions. Empty arrows indicate morning and evening peaks at CT0 and CT12 respectively. Paired student t-test was used to determine significance. w1118 (n=32). a’. Mean locomotor activity per day in controls obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). Paired student t-test was used to determine significance. w1118 (n=32) b. Representative activity profile graph of SparΔExon1 illustrating the average activity count measured every 5 min across 24 h obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). Empty arrows indicate morning and evening peaks at CT0 and CT12 respectively. Paired student t-test was used to determine significance between the two experimental conditions (****p<0.0001). SparΔExon1 (n=31). b’. Mean locomotor activity per day of SparΔExon1 obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). Paired student t-test was used to determine significance (****p<0.0001). SparΔExon1 (n=31). c. Representative average actograms of individual w1118 flies (n=32) and SparΔExon1 flies (n=31) in LD and DD conditions. Each row corresponds to one day, visualized in 288 bars each representing one 5 min interval. ZT0 and ZT12 represent the start and end of the photoperiod respectively. CT0 and CT12 represent the start and end of the subjective day in constant dark conditions. d. Average number of sleep bouts for 12 h photophase over 5 days in LD and the corresponding time over 5 days in DD. Unpaired student t-test was used to determine significance between control (w1118) and SparΔExon1 (***p<0.001). Paired student t-test was used to determine significance between the two experimental conditions (***p<0.001; **p<0.01). w1118 (n=32), SparΔExon1 (n=31). e. Representative activity profile graph of Clk856-Gal4>+ illustrating the average activity count measured every 5 min across a 24 h span for Light-Dark (LD) for 5 cycles (black line) and subsequently switching to Dark-Dark (DD) for 5 cycles (gray lines). Paired student t-test was used to determine significance (****p<0.0001). Clk856-Gal4>+ (n=32). e’. Mean locomotor activity per day of Clk856-Gal4>+ obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). Paired student t-test was used to determine significance (****p<0.0001). Clk856-Gal4>+ (n=32). Clk856-GAL4>UAS-Spar RNAi (n=27). f. Representative activity profile graph of UAS-Spar RNAi>+ illustrating the average activity count measured every 5 min across 24-hour span obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). A paired student t-test was used to determine the significance between the two experimental conditions. UAS-Spar RNAi>+ (n=32). f’. Graph illustrating the mean locomotor activity per day of UAS-Spar RNAi>+ obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). A paired student t-test was used to determine the significance between the two experimental conditions. UAS-Spar RNAi>+ (n=32). g. Representative activity profile graph of Clk856-Gal4>UAS-Spar RNAi illustrating the average activity count measured every 5 min across 24 h span obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). Paired student t-test was used to determine significance (****p<0.0001). Clk856-GAL4>UAS-Spar RNAi (n=27). g’. Mean locomotor activity per day for Clk856-Gal4>UAS-Spar RNAi obtained by averaging 5 days in light/dark conditions (LD1-LD5) and 5 days in dark/dark conditions (DD1-DD5). Paired student t-test was used to determine the significance (****p<0.0001). Error bars represent standard deviation.