Genetic network shaping Kenyon cell identity and function in Drosophila mushroom bodies
- Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan
Figures
Figure 1 with 3 supplements
Expression patterns of main Kenyon cell (KC) type markers.
(A–H) KC-type-specific GFP markers (green) were counter-stained with Trio (magenta) to reveal expression patterns at the wandering larval (WL) (A, C, E, G) and adult (B, D, F, H) stages. (A, B) Ab-GFP was primarily expressed in cell bodies of γ neurons at both WL and adult stages. The Trio signal indicates locations of γ neurons for staining observed only in cytosol (arrows) and α′/β′ neurons for staining in the entire cell (arrowheads). (C, D) Lac-FSVS expression was enriched in α′ and β′ lobes (arrowheads) of adult but not WL stage animals. The single section in the bottom panels of (D) reveals the lack of Lac-FSVS expression in the γ lobe. (E–H) E93-GFSTF and Ca-α1T-GFSTF were preferentially expressed in respective cell bodies and dendrites (the calyx) of α/β neurons (double-arrows) at adult but not WL stage animals. In addition to calyx expression, Ca-α1T-GFSTF was also seen in the protocerebral bridge (PB) of adult brains. Genotypes shown in all figures are summarized in Supplementary file 2. Scale bar: 10 µm.
Figure 1—figure supplement 1
GFP-line screen for Kenyon cell (KC) subtype markers.
Previous RNA-seq studies revealed genes of interest with the preferential expression in KC subtypes of adult brains (Alyagor et al., 2018; Shih et al., 2019). Among these genes, the expression levels of ab and mamo are enriched, respectively, in γ and α′/β′ neurons (Liu et al., 2019; Lai et al., 2022). Therefore, these markers were utilized for the identification of genes specifically expressed in KC subtypes from RNA-seq datasets (Alyagor et al., 2018; Shih et al., 2019). To leverage the RNA-seq information to obtain freely accessible reagents for studies on KC development, KC subtype marker-expressing lines were collected, each of which carry GFP transgenes either derived from engineered BAC clones or inserted in genes of interest, from fly stock centers (Venken et al., 2009; Venken et al., 2011; Morin et al., 2001). For the BAC-GFP lines, flies were generated with transgenes carrying BAC genomic DNAs of genes of interest, which contain mostly intact regulatory fragments, and an engineered DNA fragment, which permits the expression of GFP and other tags at the C-terminus of those proteins encoded by genes of interest (Venken et al., 2009). For GFP-trapping lines, flies were generated by remobilizing or inserting transgenes for expression of GFP and tags fused in frame with proteins encoded by genes of interest (Venken et al., 2011; Morin et al., 2001). One strategy utilized to generate these GFP-trapping lines is by site-specifically integrating the DNA fragment which encodes in-frame GFP and tags into a coding intron of genes of interest through the Minos-mediated integration cassette (MiMIC) system (Venken et al., 2011). To simplify this GFP-marker screen, the top list of genes enriched in γ and α/β neurons was selected using RNA-seq data from the Alyagor study to examine expression patterns (Alyagor et al., 2018). Meanwhile, all genes enriched in α′/β′ neurons from the RNA-seq data of the Shih study were examined for their capacity to serve as α′/β′-specific markers (Shih et al., 2019). In total, 7, 9, and 24 GFP lines with specific markers were, respectively, identified for γ, α/β, and α′/β′ neurons. (A–C) In addition to the selected lines described in Figure 1, the remaining GFP lines were depicted with the larval and adult expression patterns at cell body (cb) and lobe regions in Figure 1–figure supplement 1. Six (panels A1–A6), seven (panels B1–B7), and twenty-three (panels C1–C23) GFP lines were identified for potential expression in γ, α/β, and α′/β′ neurons, respectively (see Supplementary file 1 for the description of expression patterns). Of note, the Mamo-sfGFP-TVPTBF line (panel C15) did not appear to express in KCs. Trio was stained in magenta in panels. Scale bar: 10 µm.
Figure 1—figure supplement 2
Downregulation of Ab-GFP in Kenyon cells (KCs) in the chinmo mutation.
Ab-GFP expression (green) was compromised in KCs of chinmo[1] mutants in the MARCM analysis using GAL4-OK107 (white). This justifies using Ab-GFP as the replacement of Ab antibody for the readout of a γ-specific marker. Mosaic clones were induced at newly hatched larva and analyzed at adult brains. Trio (magenta) was used to label γ and α′/β′ neurons. Scale bar: 10 µm.
Figure 1—figure supplement 3
Early pupal expression of Lac-FSVS and E93-GFSTF in Kenyon cells (KCs).
(A, B) The expression of Lac-FSVS (green in panel A) and E93-GFSTF (green in panel B) was observed at 15 and 24 hr after puparium formation (APF), respectively. Lac-FSVS was expressed in α′/β′ neurons (arrowheads) according to counter-staining with cell adhesion molecule Fasciclin II (Fas2, magenta in panel A), which primarily labels γ neurons (arrows) at 15 hr APF. E93-GFSTF (double-arrows) was seen in the region with the weak RFP expression driven by GAL4-OK107 (magenta in panel B). This pattern implies that F93-GFSTF expression occurs in the newly generated KCs, which are most likely α/β neurons, at 24 hr APF. Scale bar: 10 µm.
Figure 2 with 5 supplements
E93 specifies the α/β neural identity and affects animal behaviors.
As compared to the wild-type controls (A, C), flies with overexpression of E93 RNAi (B, D) by a pan-Kenyon cell (KC) driver, GAL4-OK107, had significantly impaired expression of Ca-α1T-GFSTF and 44E04-LexA in α/β neurons (double-arrows) in adult brains. As an internal control, the PB expression of Ca-α1T-GFSTF was intact under E93 RNAi knockdown driven by GAL4-OK107. (E, F) E93 knockdown did not block the expression of 70F05-LexA in α/β neurons to cause the detectable morphological defect in lobe regions (double-arrows). (G, H) However, compared to the wild-type, Ab-GFP expression was ectopically expressed in more than half of 70F05-LexA-positive neurons (double-arrows) when E93 was knocked down in KCs. The expression levels of 44E04-LexA and 70F05-LexA were visualized by lexAop-myr-GFP in panels C-F and lexAop-mCD8::RFP in panels G and H. Lobes of γ, α/β, and α/β neurons were outlined in blue, white, and green, respectively, in panels C-F. Cell numbers of 70F05-LexA- and Ab-GFP-positive neurons were counted in Figure 2—figure supplement 4. Scale bar: 10 µm. (I) In control samples, including yw, Ca-α1T RNAi-, E93 RNAi-, and α/β-neural driver (13F02-AD/70F05-DBD)-only flies, it took around 8 hr for the minimal speed (purple spots) to be reached at night. However, flies took around 2–4 hr to achieve a minimal speed when RNAi’s for Ca-α1T and E93 were overexpressed using 13F02-AD/70F05-DBD. Moving speed (black line) from the second day to the fifth day was calculated as the overall traveling distance (mm) for 30 min. Standard deviation (in gray) for each time point is shown. The bar graph depicts the duration of food region exploration (X to Z zones, from proximal to distal). All flies, except for Ca-α1T knockdown samples, tended to explore more in the X zone in the daytime. ZT: Zeitgeber time. The setting and analysis of the behavioral assay is detailed in Figure 2—figure supplement 5.
Figure 2—figure supplement 1
Specificity of E93 RNAi reagents in blocking the expression of E93-GFSTF.
(A–C) Overexpression of either E93 RNAi [from Bloomington Drosophila Stock Center (BDSC) stock number 57868 or Vienna Drosophila Resource Center (VDRC) stock number 104390] using GAL4-OK107 (magenta) led to specific knockdown of E93-GFSTF (green) in Kenyon cells (KCs, double-arrows) in adult brains. E93 RNAi (BDSC57868) was used in the rest of this study. Scale bar: 10 µm.
Figure 2—figure supplement 2
Downregulation of Ca-α1T-GFSTF in the E93 mutation.
(A, B) As compared to the wild-type sample, the Ca-α1T-GFSTF expression (green, indicated by double-arrows) was abolished in the calyx region of E93Δ11 mutants in the MARCM analysis using GAL4-OK107 (magenta). Mosaic clones were induced in newly hatched larva and analyzed in adult brains. Scale bar: 10 µm.
Figure 2—figure supplement 3
E93 RNAi knockdown does not affect the expression of Lac-FSVS.
Compared to wild-type samples, overexpression of E93 RNAi driven by GAL4-OK107 (magenta) did not block the expression of Lac-FSVS (green) in α′/β′ neurons (arrowheads) in adult brains. Scale bar: 10 µm.
Figure 2—figure supplement 4
Statistical analysis of Ab-GFP expression in Kenyon cells (KCs) of wild-type and E93 RNAi knockdown samples in Figure 2H, I.
Compared to the wild-type (691 ± 67, n = 5), E93 RNAi knockdown driven by GAL4-OK107 did not alter the cell number of 70F05-LexA-positive neurons (682 ± 41, n = 6). However, compared to the wild-type (697 ± 24), E93 RNAi knockdown significantly increased the cell number of Ab-GFP-positive neurons (1096 ± 77). Interestingly, Ab-GFP was rarely expressed in 70F05-LexA-positive neurons in wild-type samples (6 ± 2). In contrast, Ab-GFP was expressed in more than half of all 70F05-LexA-positive neurons in E93 RNAi knockdown samples (367 ± 36). The cell numbers of Ab-GFP outside 70F05-LexA-positive neurons were similar between wild-type (691 ± 24) and E93 RNAi knockdown (728 ± 60) samples. The Student’s test was used for statistical analysis. n.s.: not significant; **: significant.
Figure 2—figure supplement 5
Setting, tracking, and related results for the behavioral assay.
(A, B) The activity monitor system was developed by DroBot, Inc. The setting of the behavioral assay was assembled by three acrylate sheets (sizes and properties as depicted). Flies were loaded using the holes on the top plate to set up left and right experimental groups. Before loading flies, food was pre-placed on distal sides of chambers. Individual fly activities were filmed for more than 4 days. Images derived from videos were analyzed by the built-in software of the DroBot activity monitor system. Average speed and standard deviation of individual flies were calculated according to total traveling distance in 30 min. (C) Samples of E93 knockdown driven by c739-GAL4 displayed curly and aberrant wings, which might compromise the general movement seen in panel E(5). (D) Mushroom body (MB) lobes appeared intact when Ca-α1T and E93 were knocked down by an α/β neural driver, 13F02-AD/70F05-DBD. (E) The overall pattern of moving speed in RNAi knockdown samples of Ca-α1T and E93 driven by GAL4-c739 (another α/β neural driver) was similar to those samples used 13F02-AD/70F05-DBD (seen in Figure 2I). Overall moving speed was lower and more variable in E93 knockdowns driven by GAL4-c739, possibly resulting from the aberrant wings seen in panel C(2). Compared to control flies, Ca-α1T and E93 knockdown flies (with GAL4-c739) explored less in the X zone, especially on day 4. ZT: Zeitgeber time. Scale bar: 0.5 mm in panel C and 10 µm in panel D.
Figure 3 with 2 supplements
E93 is sufficient to shift the Kenyon cell (KC) identity toward α/β neural-like fate.
(A, B) Overexpression of E93 driven by GAL4-OK107 caused precocious expression of α/β-specific Ca-α1T-GFSTF in early-born KCs at the wandering larval (WL) stage. (C, D) In addition, overexpression of E93 driven by a γ-neural driver, GAL4-201Y (magenta), ectopically turned on the expression of a α/β-specific 70F05-LexA driver in a portion of γ neurons (visualized by myr-GFP in green; arrow). On the other hand, overexpression of E93 abolished γ-specific markers, including Ab-GFP (E, F), MamoH/I (G, H), MamoD~G (weak green signal; I, J) and EcR-B1 (E–J), and α′/β′-specific MamoD~G (strong green signal within yellow dashed-line; I, J) in the early-born KCs at the white pupal (WP) stage. (K, L) E93 overexpression also compromised the Lac-FSVS expression in α′/β′ neurons and the morphology of mushroom body (MB) lobes revealed by cell adhesion molecule Fasciclin II (Fas2, strong magenta for labeling α and β lobes) at 24 hr after puparium formation (APF). An enhance-promoter (EP) line inserted at the proximal region of the E93-A 5′UTR was used to overexpress E93 in the gain-of-function experiments. The potency of the E93(EP) line was similar to two other in-house transgenic lines expressing E93-A and E93-B isoforms (see Figure 3—figure supplement 1). Scale bar: 10 µm.
Figure 3—figure supplement 1
E93 gene and downregulation of Ab-GFP and EcR-B1 in Kenyon cells (KCs) by overexpression of E93 isoforms.
(A) Based on the information available at Flybase, the E93 gene potentially expresses two E93 transcript variants that encode E93-A and E93-B protein isoforms. E93-A and E93-B isoforms share most of the protein sequence but differ in respective 9 and 32 unique amino acids at the N-terminus. The E93(EP) is inserted at the proximal region of the E93-A 5’UTR, was used to overexpress E93 in most of the (GOF) experiments in this paper. The E93Δ11 mutation is a small deficiency line generated by deleting the genomic DNA from exon 2 to exon 4 (Lam et al., 2022). (B, C) The expression of two γ neural-specific genes, Ab-GFP isoforms (green) and EcR-B1 (white), was almost absent in KCs at the wandering larval (WL) stage when E93-A and E93-B were overexpressed driven by GAL4-OK107 (magenta). Since similar results were found when overexpressing E93(EP), E93-A, and E93-B (Figure 3F), only the E93(EP) line was used for most of gain-of-function studies in this paper. Scale bars: 10 µm.
Figure 3—figure supplement 2
E93 overexpression using a neuroblast driver does not cause defects in Kenyon cells (KCs).
(A, B) The expression of γ neural-specific genes, Ab-GFP isoforms (green), EcR-B1 (white), and cytosolic expressed Trio (white, arrows), was intact in KCs at wandering larval (WL) and adult stages when E93(EP) overexpression was driven by a pan-neuroblast driver, Worniu (Wor)-GAL4 (magenta) (Pahl et al., 2019). Similarly, the whole-cell expression level of Trio, an α′/β′ neural-specific marker (white, arrowheads), was not altered at the adult stage by E93 overexpression. These results suggest that defects in KCs caused by E93 overexpression in Figure 3 and Figure 3—figure supplement 1 are not due to impairments in mushroom body (MB) neuroblasts. Scale bars: 10 µm.
Figure 4 with 5 supplements
Genetic networks of chinmo, mamo, E93, and ab control Kenyon cell (KC) identity.
(A, B) As compared to the wild-type, expression of Ab-GFP (green) was diminished in KCs at the first instar larval (L1) stage upon E93 overexpression driven by GAL4-OK107 (magenta). However, Chinmo expression (white) was not affected by E93 overexpression. (C–E) In contrast, RNAi knockdown of chinmo, but not mamo, driven by GAL4-OK107 (magenta) precociously turned on the expression of E93-GFSTF (green) in the early-born KCs at the wandering larval (WL) stage. (F, G) However, RNAi knockdown of mamo driven by GAL4-OK107 ectopically turned on expression of E93-GFSTF (green) in KCs with weak cytosolically expressed Trio (magenta) in adult brains (magenta dash lines). The weak Trio signal was possibly due to mamo RNAi knockdown in early-born KCs. E93-GFSTF was densely expressed in putative α/β neurons with negative Trio signal (region outside magenta dashed lines). (H–J) Ab overexpression driven by GAL4-OK107 diminished the expression of E93-GFSTF and Ca-α1T-GFSTF in KCs of adult brains. The Trio seemed to be expressed in the cytosol in almost all KCs upon Ab overexpression. Scale bar: 10 µm.
Figure 4—figure supplement 1
Precocious upregulation of E93-GFSTF in Kenyon cells (KCs) in the chinmo mutation.
E93-GFSTF expression (green) was precociously turned on in KCs of chinmo[1] mutants in the MARCM analysis using GAL4-OK107 (white). Mosaic clones were induced in newly hatched larva and analyzed at the wandering larval (WL) stage. The expression of Trio (magenta), labeling γ neurons at the WL stage, was significantly reduced in the chinmo mutant clone. Scale bar: 10 µm.
Figure 4—figure supplement 2
Overexpression of let-7 and syp RNAi compromises the expression of E93-GFSTF in Kenyon cells (KCs).
Overexpression of microRNA let-7 and RNAi of RNA-binding protein syp driven by GAL4-OK107 (magenta) led to respective upregulation and downregulation of E93-GFSTF (green) expression in KCs at wandering larval (WL) and adult stages, respectively. Scale bar: 10 µm.
Figure 4—figure supplement 3
Overexpression of Ab compromises E93-GFSTP expression in Kenyon cells (KCs).
(A–D) Overexpression of Ab (an independent transgenic line from the FlyORF stock center; stock number F000705) driven by GAL4-OK107 (white) significantly blocked E93-GFSTF expression (green) in KCs of adult brains. Similarly, as seen in Figure 4G, H, Trio (magenta) was also expressed in the cytosol in almost all KCs in FlyORF Ab gain-of-function studies. Scale bars: 10 µm.
Figure 4—figure supplement 4
RNAi knockdown of ab elicits no obvious effects on the expression of Trio, E93-GFSTF, and Ca-α1T-GFSTF in Kenyon cells (KCs).
(A–C) RNAi knockdown of ab (available at Vienna Drosophila stock center, stock number 104582) using GAL4-OK107 (magenta) specifically blocked the expression of Ab-GFP (green) in KCs at the wandering larval (WL) stage. However, ab RNAi knockdown neither abolished Trio expression (white) nor did it upregulate expression of E93-GFSTF (green) and Ca-α1T-GFSTF (green) in KCs at the WL stage. Dicer was included to increase the ab RNAi knockdown potency in experiments shown in panels A–C. Scale bars: 10 µm.
Figure 4—figure supplement 5
RNAi knockdown of ab fails to restore the Ca-α1T-GFSTF expression caused by E93 knockdown.
(A, B) RNAi knockdown of ab using GAL4-OK107 blocked the expression of Ab-GFP (green) in Kenyon cells (KCs) of adult brains. (C, D) Ca-α1T-GFSTF expression (green) was not restored in KCs with double RNAi knockdown of E93 and ab at the adult stage, while Ab-GFP upregulation caused by E93 knockdown was abolished by overexpressing ab RNAi. Dicer was included to increase the ab RNAi knockdown potency in experiments shown in panels B and D. Trio expression was unaltered in experiments shown in panels A, B, and D. 70F05-LexA-positive cells were used to test whether ab RNAi could block the upregulation of Ab-GFP expression caused by E93 knockdown in panel C. Scale bars: 10 µm.
Figure 5
Hierarchical genetic networks govern the identity and function of Kenyon cells (KCs) in the construction of Mbs.
(A, B) Scheme delineates hierarchical genetic networks among chinmo, mamo, E93, and ab with feedback loops that control the cell identities of γ and α′/β′ neurons. Syp and let-7 are included in genetic networks to potentially link the regulation of E93 and sleeping modulatory calcium channel Ca-α1T (Liu et al., 2015; Wu et al., 2012; Goodwin et al., 2018; Figure 4—figure supplement 2). Based on the results of gain-of-function studies (Figures 3H, J, 4H), possible feedback regulation in the genetic network is indicated with OE (as the abbreviation of overexpression). Since E93 regulates the Ca-α1T expression (Figure 2B) and since let-7 is also crucial for the sleep behavior (Goodwin et al., 2018), E93 and Ca-α1T may be potentially associated with sleep and memory behaviors through KCs. Question marks (?) indicate possible regulation in the genetic network.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | Ab-GFP | Bloomington Drosophila Stock Center | BDSC:38626; FLYB: FBti0147714; RRID:BDSC_38626 | FlyBase symbol: PBac{ab-GFP.FLAG}VK00033 |
| Genetic reagent (D. melanogaster) | Lac-FSVS | Kyoto Drosophila Resource Center | DGGR:115308; FLYB: FBti0143795 | FlyBase symbol: PBac{769.FSVS-1}LacCPTI002601 |
| Genetic reagent (D. melanogaster) | E93-GFSTF | Bloomington Drosophila Stock Center | BDSC:59412; FLYB: FBti0178367; RRID:BDSC_59412 | FlyBase symbol: Mi{PT-GFSTF.1}Eip93FMI05200-GFSTF.1 |
| Genetic reagent (D. melanogaster) | Ca-α1T-GFSTF | Bloomington Drosophila Stock Center | BDSC:61800; FLYB: FBti0178427; RRID:BDSC_61800 | FlyBase symbol: Mi{PT-GFSTF.0}Ca-α1TMI08565-GFSTF.0 |
| Genetic reagent (D. melanogaster) | hs-FLP[12], UAS-mCD8::GFP | Bloomington Drosophila Stock Center | BDSC:28832; RRID:BDSC_28832 | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | tubP-GAL80,FRT40A | Bloomington Drosophila Stock Center | BDSC:5192; RRID:BDSC_5192 | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | chinmo1,FRT40A | Bloomington Drosophila Stock Center | BDSC:59969; RRID:BDSC_59969 | FlyBase symbol: n.a |
| Genetic reagent (D. melanogaster) | GAL4-OK107 | Bloomington Drosophila Stock Center | BDSC:854; FLYB: FBal0242600; RRID:BDSC_854 | FlyBase symbol: eyOK107 |
| Genetic reagent (D. melanogaster) | UAS-mCD8::RFP | Bloomington Drosophila Stock Center | BDSC:32219; FLYB: FBti0131967; RRID:BDSC_32219 | FlyBase symbol: P{10XUAS-IVS-mCD8::RFP}attP40 |
| Genetic reagent (D. melanogaster) | UAS-mCD8::RFP | Bloomington Drosophila Stock Center | BDSC:32218; FLYB: FBti0131950; RRID:BDSC_32218 | FlyBase symbol: P{10XUAS-IVS-mCD8::RFP}attP2 |
| Genetic reagent (D. melanogaster) | UAS-E93-RNAiBDSC57868 | Bloomington Drosophila Stock Center | BDSC:57868; FLYB: FBti0164035; RRID:BDSC_57868 | FlyBase symbol: P{TRiP.HMC04773}attP40 |
| Genetic reagent (D. melanogaster) | UAS-E93-RNAiVDRC104390 | Vienna Drosophila Resource Center | VDRC:104390; FLYB: FBti0120934 | FlyBase symbol: P{KK108140}VIE-260B |
| Genetic reagent (D. melanogaster) | UAS-Ca-α1T-RNAi | Bloomington Drosophila Stock Center | BDSC:39029; FLYB: FBti0149691; RRID:BDSC_39029 | FlyBase symbol: P{TRiP.HMS01948}attP40 |
| Genetic reagent (D. melanogaster) | GAL4-c739 | Bloomington Drosophila Stock Center | BDSC:7362; FLYB: FBti0002926; RRID:BDSC_7362 | FlyBase symbol: P{GawB}Hr39c739 |
| Genetic reagent (D. melanogaster) | 44E04-LexA::P65 | Bloomington Drosophila Stock Center | BDSC:52736; FLYB: FBti0155872; RRID:BDSC_52736 | FlyBase symbol: P{GMR44E04-lexA}attP40 |
| Genetic reagent (D. melanogaster) | 70F05-LexA::P65 | Bloomington Drosophila Stock Center | BDSC:53629; FLYB: FBti0156295; RRID:BDSC_53629 | FlyBase symbol: P{GMR70F05-lexA}attP40 |
| Genetic reagent (D. melanogaster) | 13F02-p65.AD | Bloomington Drosophila Stock Center | BDSC:89699; FLYB: FBti0187130; RRID:BDSC_89699 | FlyBase symbol: P{R13F02-p65.AD}attP40 |
| Genetic reagent (D. melanogaster) | 70F05-GAL4.DBD | Bloomington Drosophila Stock Center | BDSC:69380; FLYB: FBti0191783; RRID:BDSC_69380 | FlyBase symbol: P{R70F05-GAL4.DBD}attP2 |
| Genetic reagent (D. melanogaster) | LexAop2-myr::GFP [VK5] | Rubin lab; Venken et al., 2009 | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | LexAop2-mCD8::RFP [attP2] | Rubin lab; Venken et al., 2009 | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | FRT82B,tubP-GAL80 | Bloomington Drosophila Stock Center | BDSC:5135; RRID:BDSC_5135 | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | FRT82B | Bloomington Drosophila Stock Center | BDSC:86313; FLYB: FBti0002074; RRID:BDSC_86313 | FlyBase symbol: P{neoFRT}82B |
| Genetic reagent (D. melanogaster) | E93Δ11 | Bloomington Drosophila Stock Center | BDSC:93128; FLYB: FBal0369310; RRID:BDSC_93128 | FlyBase symbol: Eip93FΔ11 |
| Genetic reagent (D. melanogaster) | E93(EP) | Bloomington Drosophila Stock Center | BDSC:30179; FLYB: FBti0128429; RRID:BDSC_30179 | FlyBase symbol: P{EP}Eip93FG7133 |
| Genetic reagent (D. melanogaster) | UAS- E93-A [VK37] | Yu lab; this study | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | UAS- E93-B [VK37] | Yu lab; this study | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | GAL4-201Y | Bloomington Drosophila Stock Center | BDSC:4440; FLYB: FBti0002924; RRID:BDSC_4440 | FlyBase symbol: P{GawB}Tab2201Y |
| Genetic reagent (D. melanogaster) | mamoH/I-HA | Yu lab; Venken et al., 2011 | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | MamoD~G-HA | Yu lab; Venken et al., 2011 | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | UAS-chinmo-RNAi [VK37] | Yu lab; Liu et al., 2019 | n.a. | FlyBase symbol: n.a. |
| Genetic reagent (D. melanogaster) | UAS-LUC-let7 [attp2] | Bloomington Drosophila Stock Center | BDSC:41171; FLYB: FBti0148655; RRID:BDSC_41171 | FlyBase symbol: P{UAS-LUC-mir-let7.T}attP2 |
| Genetic reagent (D. melanogaster) | UAS-syp-RNAi | Vienna Drosophila Resource Center | VDRC:33011; FLYB: FBti0098886 | FlyBase symbol: P{GD9477}v33011. |
| Genetic reagent (D. melanogaster) | UAS-mamo-RNAi | Bloomington Drosophila Stock Center | BDSC:44103; FLYB: FBti0158705; RRID:BDSC_44103 | FlyBase symbol: P{TRiP.HMS02823}attP40 |
| Genetic reagent (D. melanogaster) | UAS-ab | Bloomington Drosophila Stock Center | BDSC:23639; FLYB: FBti0077844; RRID:BDSC_23639 | FlyBase symbol: P{UAS-ab.B}55 |
| Genetic reagent (D. melanogaster) | UAS-ab-HA [ZH-86Fb] | Zurich ORFeome Project | FlyORF:000705; FLYB: FBti0161305 | FlyBase symbol: M{UAS-ab.ORF.3xHA.GW}ZH-86Fb |
| Genetic reagent (D. melanogaster) | UAS-Dcr2 | Bloomington Drosophila Stock Center | BDSC:24651; FLYB: FBti0100276; RRID:BDSC_24651 | FlyBase symbol: P{UAab S-Dcr-2.D}10 |
| Genetic reagent (D. melanogaster) | UAS-ab-RNAi | Vienna Drosophila Resource Center | VDRC:104582; FLYB: FBti0122240 | FlyBase symbol: P{KK110195}VIE-260B |
| Genetic reagent (D. melanogaster) | dan-GFP | Bloomington Drosophila Stock Center | BDSC:92324; FLYB: FBti0214343; RRID:BDSC_92324 | FlyBase symbol: P{dan-GFP.FPTB}attP40 |
| Genetic reagent (D. melanogaster) | dlp-GFSTF | Bloomington Drosophila Stock Center | BDSC:60540; FLYB: FBti0178451; RRID:BDSC_60540 | FlyBase symbol: Mi{PT-GFSTF.1}dlpMI04217-GFSTF.1 |
| Genetic reagent (D. melanogaster) | ed-GFSTF | Bloomington Drosophila Stock Center | BDSC:59777; FLYB: FBti0178373; RRID:BDSC_59777 | FlyBase symbol: Mi{PT-GFSTF.1}edMI01552-GFSTF.1 |
| Genetic reagent (D. melanogaster) | Imp-SVS | Kyoto Drosophila Resource Center | DGGR:115455; FLYB: FBti0143574 | FlyBase symbol: PBac{802.P.SVS-2}ImpCPTI003910 |
| Genetic reagent (D. melanogaster) | SIFaR-GFSTF | Bloomington Drosophila Stock Center | BDSC:60228; FLYB: FBti0178529; RRID:BDSC_60228 | FlyBase symbol: Mi{PT-GFSTF.1}SIFaRMI05376-GFSTF.1 |
| Genetic reagent (D. melanogaster) | TkR86C-GFSTF | Bloomington Drosophila Stock Center | BDSC:60549; FLYB: FBti0178567; RRID:BDSC_60549 | FlyBase symbol: Mi{PT-GFSTF.2}TkR86CMI05788-GFSTF.2 |
| Genetic reagent (D. melanogaster) | CG31637-GFSTF | Bloomington Drosophila Stock Center | BDSC:64438; FLYB: FBti0181868; RRID:BDSC_64438 | FlyBase symbol: Mi{PT-GFSTF.2}CG31637MI03598-GFSTF.2 |
| Genetic reagent (D. melanogaster) | CG43373-GFSTF | Bloomington Drosophila Stock Center | BDSC:60239 FLYB: FBti0178336; RRID:BDSC_60239 | FlyBase symbol: Mi{PT-GFSTF.0}CG43373MI05926-GFSTF.0 |
| Genetic reagent (D. melanogaster) | CG4404-GFP | Bloomington Drosophila Stock Center | BDSC:90835 FLYB: FBti0212634; RRID:BDSC_90835 | FlyBase symbol: P{CG4404-GFP.FPTB}attP40 |
| Genetic reagent (D. melanogaster) | crb-GFSTF | Bloomington Drosophila Stock Center | BDSC:61781 FLYB: FBti0178594; RRID:BDSC_61781 | FlyBase symbol: Mi{PT-GFSTF.0}crbMI05382-GFSTF.0 |
| Genetic reagent (D. melanogaster) | Lmpt-GFSTF | Bloomington Drosophila Stock Center | BDSC:66776 FLYB: FBti0185324; RRID:BDSC_66776 | FlyBase symbol: Mi{PT-GFSTF.2}LmptMI04319-GFSTF.2 |
| Genetic reagent (D. melanogaster) | mbc-SVS | Kyoto Drosophila Resource Center | DGGR:115505; FLYB: FBti0143988 | FlyBase symbol: PBac{602.P.SVS-1}mbcCPTI001082 |
| Genetic reagent (D. melanogaster) | Octbeta3R-GFSTF | Bloomington Drosophila Stock Center | BDSC:60245; FLYB: FBti0178463; RRID:BDSC_60245 | FlyBase symbol: Mi{PT-GFSTF.1}Octβ3RMI06217-GFSTF.1 |
| Genetic reagent (D. melanogaster) | Ace-GFSTF | Bloomington Drosophila Stock Center | BDSC:60260; FLYB: FBti0178684; RRID:BDSC_60260 | FlyBase symbol: Mi{PT-GFSTF.1}AceMI07345-GFSTF.1 |
| Genetic reagent (D. melanogaster) | app-GFSTF | Bloomington Drosophila Stock Center | BDSC:60283; FLYB: FBti0178413; RRID:BDSC_60283 | FlyBase symbol: Mi{PT-GFSTF.0}appMI11129-GFSTF.0 |
| Genetic reagent (D. melanogaster) | beat-IV-GFSTF | Bloomington Drosophila Stock Center | BDSC:66506; FLYB: FBti0178471; RRID:BDSC_66506 | FlyBase symbol: Mi{PT-GFSTF.1}beat-IVMI05715-GFSTF.1 |
| Genetic reagent (D. melanogaster) | Ccn-GFSTF | Bloomington Drosophila Stock Center | BDSC:60259; FLYB: FBti0178562; RRID:BDSC_60259 | FlyBase symbol: Mi{PT-GFSTF.1}CcnMI06971-GFSTF.1 |
| Genetic reagent (D. melanogaster) | CG4829-FSVS | Kyoto Drosophila Resource Center | DGGR:115623; FLYB: FBti0143506 | FlyBase symbol: PBac{810.P.FSVS-2}CG4829CPTI004450 |
| Genetic reagent (D. melanogaster) | Cyp4p3-GFSTF | Bloomington Drosophila Stock Center | BDSC:59829; FLYB: FBti0187664; RRID:BDSC_59829 | FlyBase symbol: Mi{PT-GFSTF.1}higMI05774-GFSTF.1m |
| Genetic reagent (D. melanogaster) | DAT-sfGFP | Vienna Drosophila Resource Center | VDRC:318840; FLYB: FBti0198419 | FlyBase symbol: PBac{fTRG01319.sfGFP-TVPTBF}VK00033 |
| Genetic reagent (D. melanogaster) | dnr1-GFSTF | Bloomington Drosophila Stock Center | BDSC:76236; FLYB: FBti0185341; RRID:BDSC_76236 | FlyBase symbol: Mi{PT-GFSTF.0}dnr1MI01678-GFSTF.0 |
| Genetic reagent (D. melanogaster) | dpr17-GFSTF | Bloomington Drosophila Stock Center | BDSC:61801; FLYB: FBti0178315; RRID:BDSC_61801 | FlyBase symbol: Mi{PT-GFSTF.1}dpr17MI08707-GFSTF.1 |
| Genetic reagent (D. melanogaster) | Epac-GFSTF | Bloomington Drosophila Stock Center | BDSC:66364; FLYB: FBti0183610; RRID:BDSC_66364 | FlyBase symbol: Mi{PT-GFSTF.0}EpacMI06245-GFSTF.0 |
| Genetic reagent (D. melanogaster) | eys-GFSTF | Bloomington Drosophila Stock Center | BDSC:63162; FLYB: FBti0180153; RRID:BDSC_63162 | FlyBase symbol: Mi{PT-GFSTF.2}eysMI01874-GFSTF.2 |
| Genetic reagent (D. melanogaster) | fz3-sfGFP | Vienna Drosophila Resource Center | VDRC:318166; FLYB: FBti0198654 | FlyBase symbol: PBac{fTRG00593.sfGFP-TVPTBF}VK00033 |
| Genetic reagent (D. melanogaster) | igl-GFSTF | Bloomington Drosophila Stock Center | BDSC:60527; FLYB: FBti0178491; RRID:BDSC_60527 | FlyBase symbol: Mi{PT-GFSTF.1}iglMI02290-GFSTF.1 |
| Genetic reagent (D. melanogaster) | LRP1-GFSTF | Bloomington Drosophila Stock Center | BDSC:60248; FLYB: FBti0178454; RRID:BDSC_60248 | FlyBase symbol: Mi{PT-GFSTF.1}LRP1MI06376-GFSTF.1 |
| Genetic reagent (D. melanogaster) | mamo-sfGFP | Vienna Drosophila Resource Center | VDRC:318601; FLYB: FBti0198943 | FlyBase symbol: PBac{fTRG00552.sfGFP-TVPTBF}VK00033 |
| Genetic reagent (D. melanogaster) | Mp-GFSTF | Bloomington Drosophila Stock Center | BDSC:60567; FLYB: FBti0178435; RRID:BDSC_60567 | FlyBase symbol: Mi{PT-GFSTF.0}MpMI09316-GFSTF.0 |
| Genetic reagent (D. melanogaster) | msi-GFSTF | Bloomington Drosophila Stock Center | BDSC:61750; FLYB: FBti0178348; RRID:BDSC_61750 | FlyBase symbol: Mi{PT-GFSTF.2}msiMI00977-GFSTF.2 |
| Genetic reagent (D. melanogaster) | Ndae1-GFSTF | Bloomington Drosophila Stock Center | BDSC:61778; FLYB: FBti0178493; RRID:BDSC_61778 | FlyBase symbol: Mi{PT-GFSTF.2}Ndae1MI05100-GFSTF.2 |
| Genetic reagent (D. melanogaster) | nuf-GFSTF | Bloomington Drosophila Stock Center | BDSC:61802; FLYB: FBti0178615; RRID:BDSC_61802 | FlyBase symbol: Mi{PT-GFSTF.2}nufMI09643-GFSTF.2 |
| Genetic reagent (D. melanogaster) | rhea-GFSTF | Bloomington Drosophila Stock Center | BDSC:39649; FLYB: FBti0147808; RRID:BDSC_39649 | FlyBase symbol: Mi{PT-GFSTF.0}rheaMI00296-GFSTF.0 |
| Genetic reagent (D. melanogaster) | smal-sfGFP | Vienna Drosophila Resource Center | VDRC:318203; FLYB: FBti0198848 | FlyBase symbol: PBac{fTRG00715.sfGFP-TVPTBF}VK00033 |
| Genetic reagent (D. melanogaster) | tok-GFSTF | Bloomington Drosophila Stock Center | BDSC:60550; FLYB: FBti0178631; RRID:BDSC_60550 | FlyBase Mi{PT-GFSTF.1}tokMI06118-GFSTF.1 |
| Genetic reagent (D. melanogaster) | Zasp67-sfGFP | Vienna Drosophila Resource Center | VDRC:318355; FLYB: FBti0198786 | FlyBase symbol: PBac{fTRG01384.sfGFP-TVPTBF}VK00033 |
| Antibody | anti-Fas2 (Mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat# AB_528235, RRID:AB_528235 | IF(1:100) |
| Antibody | anti-EcR-B1 (Mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat# AB_2154902, RRID:AB_2154902 | IF(1:50) |
| Antibody | anti-Trio (Mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat# AB_528494, RRID:AB_528494 | IF(1:50) |
| Antibody | anti-CD8 (Rat monoclonal) | Thermo Fisher Scientific | Cat# MCD0800, RRID:AB_10392843 | IF(1:100) |
| Antibody | anti-HA (Rat monoclonal) | Roche | Cat# 11867423001, RRID:AB_390918 | IF(1:100) |
| Antibody | anti-GFP (Rabbit polyclonal) | Thermo Fisher Scientific | Cat#: A-11122; RRID:AB_221569 | IF(1:750) |
| Antibody | anti-Chinmo (Guinea pig polyclonal) | Sokol Lab; ref #46 | Cat#: A-11122; RRID:AB_221569 | IF(1:750) |
| Antibody | anti-rabbit Alexa 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-11034, RRID:AB_2576217 | IF(1:750) |
| Antibody | anti-rat Alexa 546 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-11081, RRID:AB_25335867 | IF(1:750) |
| Antibody | anti-guinea pig Alexa 647 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-21450, RRID:AB_2534125 | IF(1:750) |
| Antibody | anti-mouse Alexa 647 (Goat polyclonal) | Jackson ImmunoResearch lab, Inc | Cat# 115-605-166, RRID:AB_2338914 | IF(1:750) |
| Chemical compound, drug | Formaldehyde 37% solution | Sigma-Aldrich | Cat# 252549 | 4% |
| Chemical compound, drug | Paraformaldehyde 16% solution | Electron Microscopy Sciences | Cat# 15710 | 4% |
| Chemical compound, drug | SlowFade Gold Antifade Mountant | Thermo Fisher Scientific | Cat# S36936 | Anti-quenching |
| Software, algorithm | LSM | Zeiss | n.a. | Image processing |
| Software, algorithm | Photoshop CS6 | Adobe | n.a. | Image processing |
| Software, algorithm | Activity monitor system | DroBot, Inc | n.a. | Behavioral analysis |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108173/elife-108173-mdarchecklist1-v1.docx
-
Supplementary file 1
Expression patterns of GFP lines in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/108173/elife-108173-supp1-v1.docx
-
Supplementary file 2
Genotypes of flies shown in each figure panel.
- https://cdn.elifesciences.org/articles/108173/elife-108173-supp2-v1.docx
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Genetic network shaping Kenyon cell identity and function in Drosophila mushroom bodies
eLife 14:RP108173.
https://doi.org/10.7554/eLife.108173.3
