Stephanie Freer

Lab Meeting 4.13.10

Post-embryonic expression of sim

• 3 anterior Sim+ clusters- DAMv1/2- BAmas1/2- Trdm

• Optic lobes • Midline of the VNC• Posterior clusters

Hartenstein (1993)

Sim

OL

D

B

T

Posterior

Anterior

Analysis of sim-GAL4 enhancer lines

To visualize the specific projection pattern for each enhancer-GAL4 line:

1. Cross enhancer-GAL4 to UAS-GFP.nls or UAS mCD8-GFP 2. Analyze embryonic and larval brains for GFP-expression in sim+ cells3. Further dissect fragments not restricted to central brain clusters

Optic lobe expression

simGFP

Laminar OL Neurons Medullar OL Neurons

Posterior central brain clusters

Lateral Posterior Cluster

simGFP

Small Posterior Cluster

Expression in the anterior clusters

BP106GFP

Intron 3-GAL4 x UAS mCD8-GFP

Intron 3 contains enhancer elements responsible for sim expression in all 3 of the anterior central brain clusters

DAMv1/2

BAmas1/2

Trdm

Further dissection of Intron 3

F2

1.4kb

F1 F3

1. Separate optic lobe expression from central brain expression capture all central brain neurons within one line

2. Generate lines for individual subsets of central brain neurons

Intron 3 F1-GAL4 x UAS mCD8-GFP

Anterior Posterior

GFP expression - a subset of anterior central brain cells - medullar optic lobe expression (subset) - posterior clusters

simGFP

DAMv1/2

BAmas1/2

DPM

Additional intron 3-GAL4 enhancer constructs

Intron 3 Frag 3

Intron 3 F2-GAL4 – no expressionIntron 3 F3-GAL4 – subset of medulla

Generated 3 500bp overlapping constructs

simGFP

F2

1.4kb

F1 F3

F1a

F1b

F1+ F2

Intron 3 F1a-GAL4 x UAS mCD8-GFP

Posterior

GFP

GFP expression - anterior central brain cell clusters - medullar optic lobe expression - lateral posterior clusters - somewhat weaker

Anterior

D

B

T

Medulla

Intron 3 F1b-GAL4 x UAS mCD8-GFP

simBP106GFP

Trdm

BAmas1/2

Intron 3 F1b GAL4 – anterior Trdm cluster - optic lobe medulla - staining weak in both cases

Intron 3 F1+F2-GAL4 x UAS mCD8-GFP

DPM

simGFP

Intron 3 F1 GAL4 expression – DAMv1/2, BAmas1/2, medulla, lateral post., DPMIntron 3 F2 GAL4 expression – unspecific cells labeled

Intron 3 F1+F2 expression – DPM, lateral post., medulla

Repressive element in intron 3 F2 fragment?

Summary of Larval Expression

 DAMv1/2, BAmas1/2

TrdmLateral

posteriorSmall

PosteriorDPM

Medulla OL

Lamina OL

Midline Other

Intron 3 GAL4 x x x   x x     x

Intron 3 F1 GAL4 x   x   x x      

Intron 3 F2 GAL4                 x

Intron 3 F3 GAL4           x      

Intron 3 F1a GAL4

x x x   x x      

Intron 3 F1b GAL4

  x       x      

Intron 3 F1+F2 GAL4

    x   x x      

3.7 sim GAL4   x           x  

1.0kb GAL4 x x x x x x x x x

Frag 2 GAL4       x     x    

Intron 3-GAL4 enhancer constructs

F2

1.4kb

F1 F3

F1a

F1b

F1+ F2

Medulla - elements throughout intron 3

DAMv1/2, BAmas1/2 – likely within 250bp F1a Trdm - within F1a, F1b, not in F1+F2Lateral posterior cluster – also likely within 250bp F1a

Post-embryonic function of sim?

1. MARCM

- sim mutants in posterior clusters

2. sim RNAi

- axon fasciculation defects

3. Adhesive/Anti-adhesive proteins

DAMv1/2 sim+ cell clusters send axons across the commissure and fasciculate together

sim²

WT

SimDAPIGFP

SimGFP

MARCM (mosaic analysis with a repressible cell marker) Generates homozygous marked/mutant cells from heterozygous precursors

Anterior MARCM GFP clones

simBP106GFP

DAMv1/2

MARCM clones in the posterior sim clusters

Posterior Anterior

simBP106GFP

simBP106GFP

3 tightly fasciculated axonal bundles cross the commissure

Posterior mutant sim

BP106DCAD2

simDCAD2

WT simH9/Df(3R)ry75

DM1 lineage

• Secondary axon tract projects toward interhemispheric region

• Enters commissure at site 1 of the DPC1, projects contralaterally, defasciculates in the neuropil

• Descending ipsilateral projection from which a small commissural bundle branches off commissural fiber bundles

WT and mutant DPM clones

simBP106GFP

WT DPM clone simH9 DPM clone

sim RNAi

• Driving UAS sim RNAi construct (+UAS dcr2) with intron 3-GAL4

• Expected: 50% ♀• All brains sim positive (n = ~40)

• Recombine UAS sim RNAi with UAS mCD8-GFP to label axons

• Reduced sim? Fasciculation defects?

DAMv1/2

BAmas1/2sim

Conclusions

Further dissection of intron 3 expression

Axonal trajectory of the lateral posterior clusters using MARCM

Defasciculation in the DPM mutant clone

Does the RNAi construct work?Is there a defasciculation phenotype when sim is reduced?

Lateral posterior cluster mutant clonesBAmas1/2 clonesTrdm clones

Axons in late sim mutant

DAMv1/2

Trdm

BAmas1/2

simBP106

Trdm?

BAmas1/2?

WT simH9/Df(3R)ry75