Stephanie Freer Lab Meeting 4.13.10
Jan 14, 2016
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