Genetic network shaping Kenyon cell identity and function in Drosophila mushroom bodies

Abstract

Revealing the molecular mechanisms underlying neuronal specification and acquisition of specific functions is key to understanding how the nervous system is constructed. In the Drosophila brain, Kenyon cells (KCs) are sequentially generated to assemble the backbone of the mushroom body (MB). Broad-complex, tramtrack, and bric-ȧ-brac zinc finger transcription factors (BTBzf TFs) specify early-born KCs, whereas the essential TFs for specifying late-born KCs remain unidentified. Here, we report that Pipsqueak domain-containing TF Eip93F promotes the identity of late-born KCs by reciprocally regulating gene expression in main KC types. Moreover, Eip93F not only regulates the expression of calcium channel Ca-α1T in late-born KCs to functionally control animal behavior, but it also forms a genetic network with BTBzf TFs to specify the identities of main KC types. Our study provides crucial information linking KC-type diversification to unique function acquisition in the adult MB.

Introduction

The nervous system contains a diverse ensemble of neuron types, which are generated during development in appropriate numbers under tightly controlled processes. A central question in developmental neurobiology concerns how diverse neuron types acquire unique cell identities with characteristic morphologies, distinct gene expression profiles, and specific functionalities. Kenyon cells (KCs) are the intrinsic neurons in the Drosophila mushroom body (MB) (Lee et al., 1999) and can serve as an excellent model system for investigations into the molecular mechanisms of neuron-type diversification. Around 2000 KCs generated by four neuroblasts are grouped into three sequentially born types, including γ, α′/β′ and α/β neurons (Lee et al., 1999). Functionally, these main KC types play different roles in short-term memory, acquisition, stabilization, and retrieval of memory (Zars et al., 2000; Krashes et al., 2007). Morphologically, both α/β and α′/β′ neurons have axons with two branches that project dorsally and medially into respective α and β, or α′ and β′ lobes, whereas γ neurons send out single axon branches that project medially into the γ lobe (Lee et al., 1999). These three types of KCs can also be distinguished by differentially expressed marker genes. For instance, broad-complex, tramtrack, and bric-ȧ-brac zinc finger transcription factor (BTBzf TF) Abrupt (Ab) is specifically expressed in γ neurons (Liu et al., 2019; Lai et al., 2022; Hu et al., 1995), whereas cell adhesion molecule Fasciclin II and Rho guanine nucleotide exchange factor Trio are expressed in different subsets of KC types (Liu et al., 2019). Therefore, specifying KC types with proper cell numbers, unique cell identities, and distinct functions is a key aspect of MB formation.

Previous studies have revealed that the other BTBzf TF, Chronologically inappropriate morphogenesis (Chinmo), exhibits a graded expression pattern in KCs and plays a crucial role in diversifying main KC types (Zhu et al., 2006). Chinmo is highly expressed in early-born γ neurons, but its expression level is low in later-born α′/β′ and absent in α/β neurons (Zhu et al., 2006). Notably, Chinmo controls the expression of the third BTBzf TF, Maternal gene required for meiosis (Mamo), functioning to specify the identities of main KC types at the larval stage via a fine-tuning process. In particular, a high expression level of Chinmo inhibits Mamo expression in γ neurons, whereas low Chinmo expression promotes Mamo production in α′/β′ neurons (Liu et al., 2019). As a gradual reduction of Chinmo expression occurs during development, Mamo, adding to its function in α′/β′ neurons, takes over Chinmo’s role of regulating the differentiation of γ neurons at the pupal stage (Liu et al., 2019; Lai et al., 2022; Zhu et al., 2006). In the absence of chinmo and mamo, molecular and morphological characteristics of γ and α′/β′ neurons are shifted toward those of α/β neurons, constituting a cell identity transformation (Liu et al., 2019; Lai et al., 2022; Zhu et al., 2006). Therefore, the late-born α/β neural identity is hypothesized to be a default status upon the loss of the neural identity determinants for early-born KCs (Liu et al., 2019; Zhu et al., 2006). However, it is also possible that key specification regulators of α/β neurons exist but have not yet been identified.

In this study, we leverage RNA-seq databases on KCs to identify type-specific markers as readouts for the cell identity of γ, α′/β′, and α/β neurons (Alyagor et al., 2018; Shih et al., 2019). By doing so, we identified a phylogenetically conserved Pipsqueak domain-containing TF, Ecdysone-induced protein 93F (Eip93F/E93), with preferential expression in α/β neurons. Loss of function of E93 not only downregulated α/β-specific gene expression, including a subunit of T-type like voltage-gated calcium channel Ca-α1T, but it also upregulated γ-specific Ab in late-born KCs, implying that an identity shift toward early-born KCs had occurred. Intriguingly, RNAi knockdown of E93 or Ca-α1T in α/β neurons further compromised animal behaviors, including foraging-related and night-time activities. In contrast, E93 overexpression precociously turned on Ca-α1T expression in early-born KCs at the expense of abolishing expression of early-born KC markers, such as Ab and Mamo. Notably, E93 was upregulated in early-born KCs in the absence of chinmo and mamo but diminished in late-born KCs upon Ab overexpression. Taken together, our results suggest that a hierarchical genetic network among chinmo, mamo, E93, and ab with potential feedback loops controls the identity and function of main KC types during the construction of functional MBs.

Results

Identification of KC-type-specific markers

By leveraging information from published RNA-seq studies showing preferentially expressed genes in adult KC types (Alyagor et al., 2018; Shih et al., 2019), we sought to identify a collection of KC-type-specific marker lines (available at stock centers) with GFP transgenes at genes of interest (Venken et al., 2009; Venken et al., 2011; Morin et al., 2001). The results of this screen for KC marker GFP lines are depicted in Figure 1—figure supplement 1 and Supplementary file 1; highlights of certain KC-type-specific marker lines are described below. First, Abrupt (Ab)-GFP, a BAC clone-engineered GFP line, can be used to replace an excellent Ab antibody (generated by Dr. Crews laboratory but no longer available) for labeling γ neurons (Liu et al., 2019; Lai et al., 2022; Hu et al., 1995; Figure 1A, B, Figure 1—figure supplement 2). In addition, Lachesin (Lac; an Ig superfamily protein; Llimargas et al., 2004)-FSVS, a GFP-trapping line, expresses GFP-fused Lac specifically in α′/β′ neurons starting from the early pupal stage (Figure 1C, D, Figure 1—figure supplement 3A). Notably, we also identified two other GFP-trapping lines, Ecdysone-induced protein 93F (E93)-GFSTF and Calcium channel protein α1 subunit T (Ca-α1T)-GFSTF, that express GFP-fused proteins enriched in α/β neurons. Enrichment in these neurons is evidenced by patterns of complementary expression to Trio, a marker of γ and α′/β′ neurons (Liu et al., 2019; Figure 1F, H). Consistent with the notion that generation of most of α/β neurons occurs at the pupal stage (Lee et al., 1999), E93-GFSTF and Ca-α1T-GFSTF were not detectable in KCs at the wandering larval (WL) stage (Figure 1E, G, Figure 1—figure supplement 3B). With this set of useful reagents, we set out to further explore the molecular mechanisms underlying cell identity specification of main KC types.

Figure 1. 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.

Loss of function of E93 suppresses the α/β-neural identity and has behavioral consequences

Since E93, a Pipsqueak-domain-containing TF, functions crucially in various biological processes (Baehrecke and Thummel, 1995; Mundorf et al., 2019; Pahl et al., 2019; Lam et al., 2022) and E93-GFSTF was preferentially expressed in α/β neurons (Figure 1F), we sought to investigate whether E93 acts as a critical regulator for specifying the α/β-neural identity. First, we found that the expression of a α/β-specific marker, Ca-α1T-GFSTF, was diminished in the context of E93 mutation (E93^Δ11) and in a line with E93 knockdown due to overexpression of E93 RNAi by a pan-KC driver, GAL4-OK107 (Lee et al., 1999; Figure 2A, B, Figure 2—figure supplements 1 and 2). In contrast, the same RNAi knockdown elicited no discernible effects on the expression levels of Trio and Lac-FSVS in γ and α′/β′ neurons (Figure 2A, B, Figure 2—figure supplement 3). In addition to the observed reduction of Ca-α1T-GFSTF, E93 knockdown in KCs substantially compromised the expression of another α/β-specific marker, myr::GFP driven by 44E04-LexA (Lai et al., 2022; Figure 2C, D). Although these results seemed to indicate the loss of α/β neural identity due to loss of E93 function, the findings also raised a possibility that the reduction of Ca-α1T-GFSTF and 44E04-LexA might simply be due to the absence of late-born KCs. However, this explanation was ruled out by the observation of relatively intact morphology of α and β lobes when we examined the expression of a third α/β-specific marker (myr::GFP driven by 70F05-LexA) upon E93 knockdown in KCs (Lai et al., 2022; Figure 2E, F). Moreover, compared to wild-type samples, the cell number of 70F05-LexA-positive neurons was not significantly altered in KCs when E93 was knocked down (Figure 2G, H, Figure 2—figure supplement 4). Taking advantage of 70F05-LexA as a putative α/β-neural marker, we further found that more than half of the 70F05-LexA-positive neurons ectopically expressed γ-specific Ab-GFP in E93 knockdown samples (Figure 2G, H, Figure 2—figure supplement 4), implying that α/β neurons had transformed into γ-like neurons. These results taken together suggested that E93, despite being unable to direct axon morphogenesis, is required for specifying KCs toward the cell identity of α/β neurons by regulating the expression of certain α/β-specific genes.

Figure 2. 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 E93^Δ11 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.

Since Ca-α1T encodes a subunit of a T-type like voltage-gated calcium channel, which regulates sleep behavior (Jeong et al., 2015), we wondered whether the loss of E93 and Ca-α1T in α/β neurons could cause behavioral defects. To examine the behavior, we utilized a monitoring system to film and analyze the activities of individual flies (Figure 2—figure supplement 5A, B). The animal’s speed of locomotion usually peaks around the day-to-night shift and gradually becomes reduced at night. In control samples (wild-type, RNAi-only, and GAL4-only lines), it took around 8 hr for the moving speed to reach a minimum during the night period (Figure 2I). Interestingly, night-time activity was significantly impaired (with a sharp reduction of moving speed to a minimal value at around 2–4 hr into the night period) when Ca-α1T and E93 were knocked down using α/β neural drivers, 13F02-AD/70F05-DBD and c739-GAL4 (Pavlowsky et al., 2024; Figure 2I, Figure 2—figure supplement 5C–E). Although the overall moving speed was lower in samples of E93 knockdown driven by c739-GAL4 than in samples of other genotypes, the wings of these E93 knockdown flies appeared curly and aberrant, which might cause the movement defect. In addition to the effects on night-time activity, RNAi knockdown of Ca-α1T and E93 seemed to compromise the foraging-related behavior (Figure 2—figure supplement 5E). Compared to control flies, Ca-α1T-deficient animals (especially for RNAi knockdown by c739-GAL4) tended to explore regions without fly food during the day-time period (Figure 2I, Figure 2—figure supplement 5E). Taken together, these results suggested that Ca-α1T (whose expression is regulated by E93) acts in α/β neurons to potentially control animal behavior.

E93 overexpression promotes α/β neural traits in early-born KCs

We next asked if E93 indeed plays a crucial role in specifying α/β neural identity, that is, does gain of E93 function transform the cell identity of other KC types to α/β neurons? In contrast to the undetectable Ca-α1T-GFSTF expression in wild-type with two main KC types, γ and α′/β′ neurons, at the WL stage, Ca-α1T-GFSTF expression was precociously upregulated in KCs when E93 was overexpressed by GAL4-OK107 (Figure 3A, B). Similarly, a portion of putative γ neurons exhibited α/β-neural like features (myr::GFP driven by R70F05-LexA) with axonal defects when E93 was overexpressed by the γ-neural driver, GAL4-201Y (Lee et al., 1999; Figure 3C, D). Consistent with this observation, we further found that E93 overexpression driven by GAL4-OK107 (but not Worniu-GAL4, a pan-neuroblast driver; Pahl et al., 2019) compromised the expression of specific markers in γ neurons at various developmental stages, including Ab-GFP, the receptor for insect molting hormone ecdysone (EcR-B1) and Mamo isoforms (Figure 3E–J, Figure 3—figure supplements 1 and 2). Along with the effect of impairing γ-specific marker expression, we further found that E93 overexpression driven by GAL4-OK107 impaired the expression of markers in α′/β′ neurons, including Mamo^D~G isoforms and Lac-FSVS (Figure 3I–L). Its overexpression also caused morphological defects by perturbing the remodeling process that normally occurs in early-born KCs at 24 hr after puparium formation (APF; Figure 3K, L). These results taken together suggested that E93, when overexpressed, is sufficient to cause specification of KCs toward the α/β neural identity.

Figure 3. 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), Mamo^H/I (G, H), Mamo^D~G (weak green signal; I, J) and EcR-B1 (E–J), and α′/β′-specific Mamo^D~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 E93^Δ11 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.

Hierarchical genetic network of chinmo, mamo, E93, and ab controls the cell identity of main KC types

Since Chinmo and E93 act as key TFs to regulate the cell identity of main KC types, we wondered whether chinmo and E93 might form a genetic network to diversify these KC types. Since chinmo controls the expression of γ-specific Ab (Liu et al., 2019; Lai et al., 2022; Figure 1—figure supplement 2), we decided to test whether the loss of Ab-GFP by E93 overexpression (seen in Figure 3F) was caused by compromised Chinmo expression. However, we did not observe a reduction of Chinmo level upon E93 overexpression at the first instar larval stage (the most abundant Chinmo expression stage in the wild-type; Zhu et al., 2006), even though the same manipulation did block Ab-GFP expression (Figure 4A, B). In contrast, E93-GFSTF expression was precociously upregulated in early-born KCs in a chinmo mutation line (chinmo^[1]) and in the chinmo knockdown line (overexpression of chinmo RNAi driven by GAL4-OK107) at the WL stage (Figure 4C, D, Figure 4—figure supplement 1). In line with the facts that microRNA let-7 and RNA-binding protein Syncrip (Syp) inhibit Chinmo expression (Liu et al., 2015; Wu et al., 2012), we further found that E93-GFSTF expression was abolished by syp RNAi knockdown and partially promoted by let-7 overexpression (Figure 4—figure supplement 2). These results suggested that the adoption of γ neural identity by early-born KCs is in part due to suppression of the α/β-neural regulator E93 via Chinmo.

Figure 4. 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.

After the diminishment of Chinmo at the early pupal stage (Zhu et al., 2006), we wonder whether E93 would be disinhibited in γ neurons and whether their neural identity would be transformed if this disinhibition indeed occurs? Since γ neurons exist in adult brains and their neural identity is crucially regulated by Mamo at the pupal stage (Lai et al., 2022), we then tested whether Mamo takes over Chinmo’s role to suppress E93 expression in γ neurons. As such, mamo RNAi knockdown indeed caused the upregulation of E93-GFSTF in KCs (within enriched Trio expression) of adult brains but not brains at the WL stage (Figure 4E–G), suggesting that Mamo could inhibit E93 expression in γ neurons to ensure their neural identity. Since the E93-mediated α/β neural identity is accompanied by absence of certain γ-specific traits, such as Ab-GFP (Figures 2H and 3F), we next sought to explore whether Ab also plays a crucial role in controlling the KC identity. Intriguingly, we found that the expression of α/β-specific Ca-α1T-GFSTF and E93-GFSTF was not detectable upon Ab overexpression (Figure 4H, J, Figure 4—figure supplement 3). This was accompanied by the expansion of Trio in the cytosol in almost all KCs, indicating the transformation of KCs into γ neuron-like cells. We also noted that RNAi knockdown of ab neither abolished Trio expression nor upregulated the expression of Ca-α1T-GFSTF caused by E93 knockdown in early-born KCs (Figure 4—figure supplements 4 and 5). Nonetheless, these results together suggested that chinmo, mamo, E93, and ab form a hierarchical genetic network with potential feedback loops to control the identity of KCs (Figure 5).

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.

Discussion

Spatially and temporally expressed regulators are employed in the developing nervous system to generate a wide diversity of neuronal types. Spatial patterning cues, such as homeodomain-containing Hox proteins and Sonic Hedgehog, are well documented in the specification of motor neurons and interneurons in the spinal cord (Jessell, 2000). Beyond spatial identity, temporal regulation provides another axis of neuronal diversification: distinct neuron types are sequentially generated to form the six-layer laminar organization of the mammalian cerebral cortex, illustrating how layer-specific neurons arise from neural progenitors according to their birth order (Kohwi and Doe, 2013; Kandel et al., 2000). Similarly, in the Drosophila central nervous system, neural progenitors (neuroblasts) produce different neuron types in an invariant sequence under the control of temporal regulators (Kohwi and Doe, 2013; Kandel et al., 2000). Distinct sets of transcription factors expressed sequentially in neuroblasts of the embryonic ventral nerve cord and developing medulla direct fate specification, thereby generating diverse neurons for larval and visual circuit assembly (Kohwi and Doe, 2013; Özel et al., 2021). In addition to sequential neuroblast regulation, temporal expression of transcription factors in postmitotic neurons also contributes to neuronal diversity by conferring distinct identities (Zhu et al., 2006; Maurange et al., 2008). For example, among the three sequentially generated KC types, γ, α′/β′, and α/β neurons, BTBzf TFs, Chinmo and Mamo, have been shown to play crucial roles in specifying early-born KCs as γ and α′/β′ neurons (Lee et al., 1999; Liu et al., 2019; Zhu et al., 2006). In this study, we revealed the molecular mechanisms of KC specification by identifying and characterizing Pipsqueak domain-containing TF E93 as a driver of KC identity toward α/β neurons. Our findings that E93 regulates the expression of calcium channel Ca-α1T to subsequently control animal behaviors also provide an illustration that neural identity specification is associated with acquisition of specific functions among main KC types. We further showed that the specification of KC types is controlled via a genetic network formed by chinmo, mamo, E93, and ab, which mainly controls the KC identity of γ and α/β neurons during construction of the adult MB (Figure 5).

Despite that reciprocal regulation between BTBzf TFs and E93 has been reported in multiple cell types during the Drosophila development (Truman and Riddiford, 2022; Cruz et al., 2024), here, we disclosed a novel strategy of using genetic networks of BTBzf TFs and E93 to specify distinct cell identities among neurons derived from the same neuroblasts (Figure 5). To diversify KC types, Chinmo and Mamo take turns to inhibit the E93 expression, thereby establishing the cell identity of γ neurons, as suggested by Ab expression (Liu et al., 2019; Figures 2H and 4G). Without the inhibitory effects of Chinmo and Mamo on E93 expression, late-born KCs specify into α/β neurons by turning on the expression of α/β-specific genes and suppressing the expression of γ-specific genes, Ab included (Figure 2). Since E93 is an ecdysone-induced protein and Mamo expression is primed by ecdysone signaling in γ neurons (Lai et al., 2022; Baehrecke and Thummel, 1995), blocking EcR-B1 is expected to enhance E93 expression in these neurons. By the fact that overexpression of E93 inhibited EcR-B1 expression in γ neurons (Figure 3F, H, J), future investigations should be able to unravel the intricate regulatory interplay between E93 and ecdysone signaling. In addition, since E93 knockdown did not alter the lobe morphology of α/β neurons (Figure 2F), it is also possible that unidentified regulators might work in concert with E93 to specify the cell fate of α/β neurons. Although how the genetic network of BTBzf TFs and E93 specifies α′/β′ neural identity has not yet been fully resolved, the Mamo expression promoted by the low level of Chinmo, which is regulated by the transforming growth factor beta signaling in MB neuroblasts, is crucial for KCs to adopt the α′/β′ neural identity (Liu et al., 2019; Rossi and Desplan, 2020). Intriguingly, our results revealed that E93 overexpression can abolish the expression of Mamo to block KCs from adopting the α′/β′ neural identity (Figure 3J, L). All these results taken together portray that the genetic network of BTBzf TFs and E93 is very likely to control the KC identity during construction of the adult MB. In light of the phylogenetic conservation of the E93 family and tramtrack (TTK)-type BTBzf TFs, including chinmo, mamo, and ab, as Arthropoda-specific genes (Bonchuk et al., 2024; Takayanagi-Kiya et al., 2017), one cannot help but wonder whether TTK-type BTBzf TFs might have been evolutionarily introduced to intervene with E93 to regulate the development of multiple arthropodan tissues; perhaps future investigations may delineate their relationship on neuron-type diversification in the nervous system.

Once the identities of neuron types are determined by key regulators, how do the cells acquire specific functions in the nervous system to eventually participate in animal behaviors? Previous findings, together with our current studies, might shed some light on this topic. First, the expression of Chinmo and Ab is inhibited in late-born KCs, in part due to the preferential expression of let-7 in α/β neurons (Wu et al., 2012; Kucherenko et al., 2012). In addition, the neuronal (and KC) contribution of let-7 regulates day- and night-time sleeping behaviors in developmental- and adult-restricted manners (Goodwin et al., 2018). Furthermore, Chinmo and let-7 potentially regulate the expression of E93, for which expression in α/β neurons governs the expression of a phylogenetically conserved sleep-modulatory calcium channel Ca-α1T to regulate animal behaviors (Jeong et al., 2015; Figures 2B and 4D, Figure 4—figure supplement 2A). Interestingly, blocking neurotransmission with tetanus toxin light chain (TeNT-Ln) in the MB has been shown to promote locomotion, an outcome opposite to the Ca-α1T knockdown results observed in our study (Martin et al., 1998; Figure 2I, Figure 2—figure supplement 5E). This discrepancy may stem from TeNT-Ln acting on MB axons to inhibit downstream neurons, whereas the effect of Ca-α1T knockdown primarily occurs in MB dendrites that receive inputs from upstream neurons (Martin et al., 1998; Figure 2I). These results, together with the previously reported function of E93 in circadian rhythm, provide an intriguing link between E93 and sleep-associated behaviors (Yip et al., 2024). Since KC types were reported to function crucially in sleep/arousal and alcohol-induced sleep deficit behaviors (Sengupta et al., 2019; Draper et al., 2024; Chvilicek et al., 2025), our finding of KC identity specification and Ca-α1T expression under the regulation of E93 suggests that this process may in some way endow α/β neurons with the capacity to control animal behaviors related to sleep. Since the intricate relationship between sleep and memory is well established (Walker et al., 2002; Graves et al., 2003; Seugnet et al., 2008; Li et al., 2009), future deeper investigations on cell-type specification of memory-associated neurons are expected to provide insights into how to acquire unique functions among neurons to regulate these two crucial traits of animals.

Materials and methods

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

Experimental model and subject details

Flies were cultured in a room maintained at 25°C (±1.5°C) and 50–65% humidity for all experiments. For most experiments, flies were used with no selection for sex; therefore, roughly equal numbers of males and females were used. However, since homozygous Ca-α1T-GFSTF females and hemizygous Ca-α1T-GFSTF males are relatively feeble, heterozygous Ca-α1T-GFSTF females were selected for the analysis in Figures 1G, H—4I, J, Figure 2—figure supplement 2, Figure 4—figure supplements 3 and 4 due to the cytolocation of the Ca-α1T gene on the X chromosome. In addition, males were selected for the behavioral assay to mitigate complications of egg laying behaviors on locomotion tracking.

Fly strains

The fly strains used in this study were as follows. Most strains are available from either Bloomington Drosophila stock center (BDSC), Kyoto Drosophila stock center (DGGR), or Vienna Drosophila stock center (VDRC). (1) Ab-GFP (BDSC38626); (2) Lac-FSVS (DGGR115308); (3) E93-GFSTF (BDSC59412); (4) Ca-α1T-GFSTF (BDSC61800); (5) hs-FLP¹²,UAS-mCD8::GFP (BDSC28832); (6) tubP-GAL80,FRT^40A (BDSC5192); (7) chinmo^[1],FRT^40A (BDSC59969); (8) GAL4-OK107 (BDSC854); (9) UAS-mCD8::RFP [attp40] (BDSC32219); (10) UAS-mCD8::RFP [attp2] (BDSC32218); (11) UAS-E93 RNAi (BDSC57868); (12) UAS-E93 RNAi (VDRC104390); (13) UAS-Ca-α1T RNAi (BDSC39029); (14) GAL4-c739 (BDSC7362); (15) 44E04-LexA::P65 (BDSC52736); (16) 70F05-LexA::P65 (BDSC523629); (17) lexAop2-myr::GFP (Pfeiffer et al., 2008); (18) lexAop2-mCD8:: RFP (Pfeiffer et al., 2008); (19) FRT^82B,tubP-GAL80 (BDSC5135); (20) FRT^82B (BDSC86313); (21) E93^Δ11 (BDSC93128); (22) E93(EP) (BDSC30179); (23) UAS-E93-A [VK37] (this study); (24) UAS-E93-B [VK37] (this study); (25) GAL4-201Y (BDSC4440); (26) mamo^H/I-HA (Chu et al., 2024); (27) mamoD ^D~G-HA (Chu et al., 2024); (28) UAS-chinmo RNAi [VK37] (Lai et al., 2022); (29) UAS-LUC-let7 (BDSC41171); (30) UAS-Ssyp RNAi (VDRC33011); (31) UAS-mamo RNAi (BDSC44103); (32) UAS-ab (BDSC23639); (33) UAS-ab-HA (FlyORF000705); (34) UAS-Dcr2 (BDSC24651); (35) UAS-ab RNAi (VDRC104582); (36) 13F02-p65.AD (BDSC68291); (37) 70F05-GAL4.DBD (BDSC69380). The UAS-E93-A and UAS-E93-B transgenes were generated using standard molecular biology methods to clone cDNA fragments derived from fully sequenced EST clones, GH10557 and LP08695 (available from Drosophila Genomics Resource Center), carrying E93-A and E93-B isoforms into the attB-UAST vector. The generation of UAS-E93-A and UAS-E93-B transgenes and fly strains was performed by WellGenetics, Inc.

RNAi knockdown and overexpression experiments and MARCM clonal analyses

UAS-RNAi and UAS-transgene lines were crossed to GAL4-107 and GAL4-201Y for knockdown and overexpression of genes of interest in KCs. Mosaic clones for the MARCM studies were generated as previously described (Lee and Luo, 1999). In short, mosaic clones of chinmo^[1] and E93^Δ11 mutations were induced by 35 min of heat shock using hs-FLP^[12] in newly hatched larva. Dissection, immunostaining, and mounting of adult brains were performed as described in a standard protocol (Lee and Luo, 1999). Primary antibodies used in this study included guinea pig antibody against Chinmo (1:1000, Sokol laboratory Chawla et al., 2016), rat monoclonal antibody against mCD8 (1:100, Thermo Fisher Scientific), rabbit antibody against GFP (1:750, Thermo Fisher Scientific), and mouse monoclonal antibodies against EcR-B1 (1:50, DSHB), Fas2 (1:100, DSHB) and Trio (1:50, DSHB). Secondary antibodies conjugated to different fluorophores (Alexa 488, Alexa 546, and Alexa 647; Thermo Fisher Scientific and Jackson ImmunoResearch Lab, Inc) were used at 1:750 dilutions. Immunofluorescence images were collected by confocal microscopy on a Zeiss LSM 700, projected using the LSM browser and processed in Adobe Photoshop CS6. All data are representative of more than 3 brains per genotype.

Distance measurement in the behavioral assay and statistical analysis

Individual fly activities were recorded and analyzed using the activity monitor system developed by DroBot, Inc. This system used an open-resourced software pySolo to track individual moving flies (Gilestro and Cirelli, 2009). Average speed and standard deviation of individual flies were calculated according to total traveling distance within 30 min periods from the first day to the fifth day, as shown in Figure 2I, Figure 2—figure supplement 5E. Student’s t-test was used for statistical analysis to compare datasets with two groups in Figure 2—figure supplement 4.

Funding Statement

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

• National Science and Technology Council NSTC-112-2311-B-001-029 to Hung-Hsiang Yu.

• Academia Sinica AS-TP-113-L01 to Hung-Hsiang Yu.

Data availability

Dataset uploaded to Dryad at: https://doi.org/10.5061/dryad.7wm37pw7n.

The following dataset was generated:

Yu HH. 2026. Genetic network shaping Kenyon cell identity and function in Drosophila mushroom bodies. Dryad Digital Repository.