Expression patterns of KC subtype markers.

(A-H) KC subtype-specific GFP markers (green) were counter-stained with Trio (magenta) to reveal expression patterns at the wandering larval (WL) (A, C, E and G) and adult (B, D, F and 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 Fig. 1d reveal 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 Table 1. Scale bar: 10 µm.

E93 specifies the α/β neural identity and affects animal behaviors.

As compared to the wild-type controls (A and D), flies with overexpression of E93 RNAi (B and E) by a pan-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. (C) In control samples, including yw, Ca-α1T RNAi-, E93 RNAi– and GAL4-c739 (an α/β neural driver)-only flies, it took around eight hours for the minimal speed (purple spots) to be reached at night. However, flies took around two hours to achieve a minimal speed when RNAi’s for Ca-α1T and E93 were overexpressed using GAL4-c739. Overall moving speed was lower and more variable in E93 knockdowns. Moving speed (black line) was calculated as the overall traveling distance (mm) for 30 min. Standard deviation (in grey) 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 tended to explore more in the X zone in later days. Compared to control flies, Ca-α1T and E93 knockdown flies explored less in the X zone, especially on day four. ZT: Zeitgeber time. The setting and analysis of the behavioral assay is detailed in S7 Fig. (F-G) 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). (H-I) 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 d-g and lexAop-mCD8::RFP in panels H and I. Cell numbers of 70F05-LexA– and Ab-GFP-positive neurons were counted in S8 Fig. Scale bar: 10 µm.

E93 is sufficient to shift the KC identity towards 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 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). (E-J) 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 MB lobes revealed by cell adhesion molecule Fasciclin II (Fas2, strong magenta for labeling α and β lobes) at 24 h 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 S9 Fig). Scale bar: 10 µm.

Genetic networks of chinmo, mamo, E93 and ab control 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 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. Trio seemed to be expressed in the cytosol in almost all KCs upon Ab overexpression. Scale bar: 10 µm.

Hierarchical genetic networks govern the identity and function of Kenyon cell 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 [ref. #20, #21 #and 27] (S12 Fig). Based on the results of gain-of-function studies (Fig 3H-3J and 4H), possible feedback regulation in the genetic network is indicated with OE (as the abbreviation of overexpression). Since E93 regulates the Ca-α1T expression (Fig 2B) and since let-7 is also crucial for the sleep behavior [ref. #28], E93 and Ca-α1T may be potentially associated with sleep and memory behaviors through KCs. Question marks (?) indicate possible regulation in the genetic network.