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

s11

s10

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?

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

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?)

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 (?)

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

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

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?)

Wingless Expression

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

wg s10

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)

Wingless Misexpression and Transplantation

wg transplantation– Inhibits l(1)sc expression

wg misexpression– No affect on en expression– No affect on axon scaffold

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?

Vein Expression

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

s6 s10 s11

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

Spitz-Vein Double Mutants

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

vn spi/+

s16 anti-Sim

wt

spi

vn spi

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)