Figure 9. Ia-2 and Dilp-6 induced ectopic neural stem cells can divide.

All samples were analysed at 120 hr AEL, after disappearance of abdominal developmental neuroblasts. (A–C’ and E–G’) Cell proliferation was visualised with the S-phase marker PCNA-GFP, quantification in (D and H). dilp-6 expression was induced in all cells with heat-shock-GAL4, raising the temperature to 37°C for 30 min at the end of the third instar larval stage at 110.5 hr AEL, and then larvae were kept at 25°C for 9 hr, visualising Dpn+ and PCNA-GFP at 120 hr AEL. (A–C’) Overexpression of dilp-6 resulted in Dpn+ PCNA-GFP+ cells laterally around the neuropile (B and B’ white arrows) and along the midline (C and C’ yellow arrowheads), showing that these ectopic Dpn+ cells were in S-phase. Quantification box-plots in (D), Student's t-test. There were also some Dpn+ cells that were not dividing (white arrows in C’). (E–G’) Overexpression of dilp-6 resulted in PCNA-GFP+ Wrp+ midline glia (yellow arrowheads) that therefore were dividing. In (G) there is a notable increase in the number of Wrp+ cells. In (E and E’) lateral PCNA-GFP+Wrp⁻Dpn+ cells around the neuropile (white arrows) most likely correspond to neuropile glia. (H) Quantification showing phenotypic penetrance: percentage of segmentally repeated Wrp+ cell clusters that contain PCNAGFP+ cells. Fisher’s exact test p=0.0276. (I–J’) Overexpression of either dilp-6 or ia-2 with hsGAL4 upregulated the S-phase marker PCNA-GFP in Wrp+ Dpn+ midline cells, meaning these ectopic Dpn+ cells were dividing. Penetrance: >dilp-6 25% N = 4; >ia-2: 18% N = 11 ventral nerve cords (VNCs). (K and K’) Overexpression of ia-2 in glia with repoGAL4 induced non-autonomously proliferation of ectopic Wrp+ Dpn+ cells, visualised with the mitotic marker pH3. Penetrance: 60% N = 10 VNCs.