Hedgehog Expression Hedgehog Expression in posterior of segment

Hedgehog Expression Ø Hedgehog – Expression in posterior of segment (s 6 -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 s 10 then in ectoderm until 11 s 10 s 11

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

Hedgehog Mutants Ø hh mutant (Bossing) – l(1)sc absent » all (? ) – – early en (s 9) ok Late en absent – sim expression reduced – – ts: hh functions at s 10 -12 Some midline cells die by stage 17

Hedgehog Mutant Ø hh mutant (Klämbt) – Increase in AMG (extra MG) at expense of neurons – Conc. : PMG → AMG, effects midline neuron development AA 142: wild-type AA 142: hh (increase in AMG) Reduced X 55: (PMG? ) AA 142: ptc (reduced AMG) High X 55: (PMG? )

Hedgehog Misexpression and Transplantation Ø hh misexpression (sim-Gal 4 UAS-hh) – Induces l(1)sc and en expression in all midline cells – Anterior commissure absent; posterior commissure present – Odd+ MP 1 s are absent and MG disrupted (? )

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

Engrailed Misexpression Ø en misexpression – Reduction in MP 1 s and MG – MG differentiation affected – Eliminates anterior commissure – Similar to hh function – Posterior neurons relatively present and relatively normal

Model Ø Model – hh subdivides segment into anterior and posterior compartments – In posterior: hh activates l(1)sc expression (release of Ci 76) then en (via l(1)sc or Ci-long) – l(1)sc and En are required formation of VUMs and MNB – lack of hh signaling in anterior gives rise to MP 1 s and AMG Ø Ø Ø lack of hh signaling results in MP 1 group Presence of hh signaling results in MP 4 group MP 3 group? (wingless? )

Wingless Expression Ø wg misexpression (Bossing; Wheeler) – wg expressed anterior to En+ and hh+ cells – On at stage 10, later (? ) wg s 10

Wingless Mutant Ø wg mutant (Bossing) – Increase l(1)sc cells from 6 → 10 – Reduction to 3 -5 En+ cells (only early En, not later En) Ø wg mutant (Klämbt) – Increased AA 142+ MG 3 → 10 cells – Neuronal markers may also be present (mixed cells)

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

Wingless Model Ø Ø Ø 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 AA 142+ 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?

Vein Expression Ø vein expression – All midline cells from stages 7 -10 – Stage 11: subset (AMG? ) – Stage 12: off s 6 s 10 s 11

Vein Mutants Ø vein mutant – wt: st 11: 6 AA 142+ cells; st 17: 3 AA 142+ cells – vein: st 12: 3 AA 142+ cells; st 17: 3 AA 142+ cells – vein: early role in generating AMG – st 17: axon scaffold normal; disorganized earlier s 12 s 17 wt vn

Spitz-Vein Double Mutants s 16 anti-Sim Ø 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

Midline Glia Pro-apoptotic Genes Ø Ø Expression – hid expression high in AMG, PMG(? ) – rpr expression transient; detected in small # cells, AMG or PMG (? ) – grim expression (? ) Genetics – slit-lac. Z s 16 » wt: 3 MG » hid mutant: 6 MG (AMG, no PMG) • spi activation results in phosphorylation of hid and inhibition of apoptosis » H 99 (hid- rpr- grim-): 10 MG (AMG + PMG); grim and rpr required for PMG » X 25 (hid- grim-): 8 MG (AMG + 1 -2 PMG) » Model: hid (AMG); grim + rpr (PMG)