Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila

A–E

Representative images showing that both pan‐glial (repo‐GAL4>) and cortex glial (NP2222‐GAL4>) htl^ACT overexpression significantly reduce NB EdU index, quantified in (E) (n = 15, 14; 10, 16 brain lobes). (A, B, C, D) are single sections with Mira and EdU staining, and (A’, B’ C’, D’) are Z‐projection of the EdU staining.

F–H

Representative still images from ex vivo CNS live imaging at 72ALH showing that pan‐glial (repo‐GAL4) htl^ACT overexpression lengthens NB cell cycle, quantified in (F) (n = 25, 6 NBs imaged from three brains per genotype). The cell cycle length is measured as the length between consecutive divisions. NBs (Dpn::GFP, red; Histone RFP, grey) are circled with blue dashed lines.

I

Schematic depicting concurrent glial FGF activation (repo‐LexA > LexAop‐htl^ACT), and NB overexpression of fly‐FUCCI (dnab‐GAL4 > UAS‐GFP::E2F1, UAS‐RFP::CycB). The fly‐FUCCI system utilises the fusion protein GFP::E2F1 (a marker for cells in G2, M and G1 phase) and RFP::CycB ( a marker for cells in S, G2, M phase) to monitor cell cycle progression. Cells in G1 phase are GFP⁺ RFP^‐ (green), cells in G2/ M phase are GFP⁺RFP⁺ (yellow), and cells in S phase are GFP^‐RFP⁺ (red), whereas cells in G1‐S transition are weakly labelled by both GFP and RFP (grey).

J–L

Representative images showing that the percentage of NBs in G1‐S transition and G2/M phase are both significantly increased with significantly less cells remaining in G1 phase, quantified in (L) (n = 9, 10 brain lobes). NBs in G1 phase (Mira⁺, GFP⁺) are circled by green dashed lines; NBs in G1‐S transition (Mira⁺, GFP^‐RFP^‐) are circled by grey dashed lines; NBs in S phase (Mira⁺, RFP⁺) are circled by red dashed lines, and NBs in G2/M phase (Mira⁺, GFP⁺RFP⁺) are circled by yellow dashed lines.

M–O

Representative images showing that the number of EdU⁺ neurons generated per NB is significantly reduced upon pan‐glial overexpression of FGF (repo‐LexA > LexAop‐htl^ACT; yellow arrows), quantified in (O) (Box plot, the boxes extend from the 25^th to 75^th percentiles; the median is marked by a central band inside the box; and the whiskers go down to the minimum value and up to the maximum value. n = 94, 127 NB lineages imaged from five and seven brain lobes, respectively). NB lineages are marked with dnab‐gal4 > GFP.

Figure 5. Cortex glial overgrowth mediated by htlACT overexpression triggers NB cell cycle delay.

Figure 5. Cortex glial overgrowth mediated by htl^ACT overexpression triggers NB cell cycle delay.