Figure 4

Newborn Kenyon cells express high levels of Glu in the Drosophila mushroom body in late larval stage and during pupal development. (A, B) Transverse section of the MB pedunculus from 201Y-GAL4 (A) and 17d-GAL4 (B) pharate adults collected a few hours before eclosion and expressing mCD8::GFP. (A1-A3) GFP and Glu expression in 201Y-GAL4 are located both in the α/βc and γ lobes. Most of the GFP-positive α/βc neurons do not co-localize with Glu and only few axons express both Glu and GFP at the border of Glu-containing a/βc neurons. Agarose section of the pedunculus close to the lobes. (B1-B3) In 17d-GAL4, most Glu-containing α/βc neurons do not express GFP and only the border axons are both GFP- and Glu-positive. Agarose section of the pedunculus close to the calyx. In (A, B), Glu immunoreactivity in the γ lobe most likely originates from extrinsic neurons. (C-I) Glu immunoreactivity monitored at late larval and pupal stages in the MB of wild-type Drosophila. (C, D). Transverse sections through the calyx and lobes of wandering third instar larva. The four bundles of Glu-positive processes originate from four clusters of KCs. Only one cluster is shown (on the left side in (C)) and arrows show bundles originating from the other Glu-positive clusters. The four Glu-positive bundles extend in the core area of the pedunculus (arrowheads), indicating that they correspond to newborn KCs. As shown in (D), these Glu-positive fibers also project into the α/βc region of the larval MB (βc is not visible on this agarose section). The contour indicates the shape of the larval MB vertical lobe (α) and spur (sp) region. Note that extrinsic glutamatergic cells are also present at this stage in the spur area. (E) In pupa 48 hours after puparium formation (APF) at 22°C (stages P5 to P6 of Bainbridge and Bownes [100]), clusters of Glu-positive KCs are still present but their bundles at the base of the pedunculus (arrowheads) are less intensely labeled compared to larval and other pupal stages. (F, G) Pupa 72 hours APF (stage P8). At this developmental stage, four groups of newborn KCs are brightly Glu-positive and project axons in the pedunculus (arrowheads in (G)). Other Glu-immunoreactive processes in the γ lobe area of the pedunculus likely correspond to fibers from extrinsic cells as only cell bodies of newborn KCs express high Glu-immunoreactivity. (H, I) In pupa 96 hours APF at 22°C (stages P11 to P12), four clusters of Glu-immunopositive newborn KCs (arrows in (H)) project their axons to the pedunculus (arrowheads in (I)). K, Kenyon cell bodies; ca, calyx, ped, pedunculus; sp, spur. Scale bars: 10 μm.