1. Hedgehog Expression • Hedgehog • Expression in posterior of segment (s6-11) • Wheeler: stage 9 in posterior of segment; very weak (or absent) at stages 10-11 in midline but present adjacent to midline • Bossing protein in midline until s10 then in ectoderm until 11 s10 s11

2. Patched Expression • Patched or Ptc-lacZ • Present in l(1)sc+ En+ cells at stage 10 • Is ptc adjacent to or includes hh+ cells? • When does ptc first appear? • Is ptc present in hh mutants?

3. Hedgehog Mutants • hh mutant (Bossing) • l(1)sc absent • all (?) • early en (s9) ok • Late en absent • sim expression reduced • ts: hh functions at s10-12 • Some midline cells die by stage 17

4. Hedgehog Mutant • hh mutant (Klämbt) • Increase in AMG (extra MG) at expense of neurons • Conc.: PMG → AMG, effects midline neuron development AA142: wild-type AA142: hh (increase in AMG) Reduced X55: (PMG?) AA142: ptc (reduced AMG) High X55: (PMG?)

5. Hedgehog Misexpression and Transplantation • hh misexpression (sim-Gal4 UAS-hh) • Induces l(1)sc and en expression in all midline cells • Anterior commissure absent; posterior commissure present • Odd+ MP1s are absent and MG disrupted (?)

6. Lethal(1) Scute • l(1)sc expression • Overlaps En+ cells • l(1)sc mutant • En expression reduced (similar to hh) • l(1)sc misexpression • No effect on en expression

7. Engrailed Misexpression • en misexpression • Reduction in MP1s and MG • MG differentiation affected • Eliminates anterior commissure • Similar to hh function • Posterior neurons relatively present and relatively normal

8. Model • Model • hh subdivides segment into anterior and posterior compartments • In posterior: hh activates l(1)sc expression (release of Ci76) then en (via l(1)sc or Ci-long) • l(1)sc and En are required for formation of VUMs and MNB • lack of hh signaling in anterior gives rise to MP1s and AMG • lack of hh signaling results in MP1 group • Presence of hh signaling results in MP4 group • MP3 group? (wingless?)

9. Wingless Expression • wg misexpression (Bossing; Wheeler) • wg expressed anterior to En+ and hh+ cells • On at stage 10, later (?) wg s10

10. Wingless Mutant • wg mutant (Bossing) • Increase l(1)sc cells from 6 → 10 • Reduction to 3-5 En+ cells (only early En, not later En) • wg mutant (Klämbt) • Increased AA142+ MG 3 → 10 cells • Neuronal markers may also be present (mixed cells)

11. Wingless Misexpression and Transplantation • wg transplantation • Inhibits l(1)sc expression • wg misexpression • No affect on en expression • No affect on axon scaffold

12. Wingless Model • wg maintains early en expression • wg inhibits anterior spread of hh signaling • Inhibits l(1)sc and early en+ cells anterior cells • Predict that wg mutant would result in fewer AA142+ cells, but instead they increase • Maybe loss of early En results in more anterior fates that cause formation of more AMG • How do Notch, Sim, Egfr, Vein, Spitz fit?

13. Vein Expression • vein expression • All midline cells from stages 7-10 • Stage 11: subset (AMG?) • Stage 12: off s6 s10 s11

14. Vein Mutants • vein mutant • wt: st11: 6 AA142+ cells; st17: 3 AA142+ cells • vein: st 12: 3 AA142+ cells; st17: 3 AA142+ cells • vein: early role in generating AMG • st17: axon scaffold normal; disorganized earlier s12 s17 wt vn

15. Spitz-Vein Double Mutants s16 anti-Sim • vein and spi single mutants • No effect on ventral-most midline cells (neurons) • spi vn double mutant: strong reduction in neurons • More severe scaffold defects • vn expression requires spi wt spi vn spi/+ vn spi

16. Midline Glia Pro-apoptotic Genes • Expression • hid expression high in AMG, PMG(?) • rpr expression transient; detected in small # cells, AMG or PMG (?) • grim expression (?) • Genetics • slit-lacZ s16 • wt: 3 MG • hid mutant: 6 MG (AMG, no PMG) • spi activation results in phosphorylation of hid and inhibition of apoptosis • H99 (hid- rpr- grim-): 10 MG (AMG + PMG); grim and rpr required for PMG • X25 (hid- grim-): 8 MG (AMG + 1-2 PMG) • Model: hid (AMG); grim + rpr (PMG)