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Stephanie Freer Lab Meeting 4.13.10 Post-embryonic expression of sim Hartenstein (1993) Posterior D OL B T Anterior Sim • 3 anterior Sim+ clusters - DAMv1/2 - BAmas1/2 - Trdm • Optic lobes • Midline of the VNC • Posterior clusters 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+ cells 3. Further dissect fragments not restricted to central brain clusters Optic lobe expression sim GFP Laminar OL Neurons Medullar OL Neurons Posterior central brain clusters sim GFP Small Posterior Cluster Lateral Posterior Cluster Expression in the anterior clusters DAMv1/2 BAmas1/2 Trdm BP106 GFP 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 Further dissection of Intron 3 1.4kb F1 F3 F2 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 DPM DAMv1/2 BAmas1/2 sim GFP Anterior Posterior GFP expression - a subset of anterior central brain cells - medullar optic lobe expression (subset) - posterior clusters Additional intron 3-GAL4 enhancer constructs 1.4kb F1 F3 F2 F1a F1b F1+ F2 Generated 3 500bp overlapping constructs Intron 3 F2-GAL4 – no expression Intron 3 F3-GAL4 – subset of medulla sim GFP Intron 3 Frag 3 Intron 3 F1a-GAL4 x UAS mCD8-GFP D B Medulla T GFP Anterior Posterior GFP expression - anterior central brain cell clusters - medullar optic lobe expression - lateral posterior clusters - somewhat weaker Intron 3 F1b-GAL4 x UAS mCD8-GFP BAmas1/2 Trdm Intron 3 F1b GAL4 – anterior Trdm cluster - optic lobe medulla - staining weak in both cases sim BP106 GFP Intron 3 F1+F2-GAL4 x UAS mCD8-GFP DPM sim GFP Intron 3 F1 GAL4 expression – DAMv1/2, BAmas1/2, medulla, lateral post., DPM Intron 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 Trdm Lateral posterior Intron 3 GAL4 x x Intron 3 F1 GAL4 x Small Posterior DPM Medulla OL x x x x x x Lamina OL Midline x Intron 3 F2 GAL4 x Intron 3 F3 GAL4 Intron 3 F1a GAL4 x x Intron 3 F1b GAL4 x x x x x 3.7 sim GAL4 Frag 2 GAL4 x x Intron 3 F1+F2 GAL4 1.0kb GAL4 Other x x x x x x x x x x x x x x x Intron 3-GAL4 enhancer constructs 1.4kb F1 F3 F2 F1a F1b F1+ F2 Medulla - elements throughout intron 3 DAMv1/2, BAmas1/2 – likely within 250bp F1a Trdm - within F1a, F1b, not in F1+F2 Lateral 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 Sim GFP Sim DAPI GFP WT sim² MARCM (mosaic analysis with a repressible cell marker) Generates homozygous marked/mutant cells from heterozygous precursors Anterior MARCM GFP clones DAMv1/2 sim BP106 GFP MARCM clones in the posterior sim clusters Posterior Anterior sim BP106 GFP sim BP106 GFP 3 tightly fasciculated axonal bundles cross the commissure Posterior mutant sim simH9/Df(3R)ry75 WT sim DCAD2 BP106 DCAD2 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 WT DPM clone simH9 DPM clone sim BP106 GFP sim RNAi • Driving UAS sim RNAi construct (+UAS dcr2) with intron 3-GAL4 • Expected: 50% ♀ • All brains sim positive (n = ~40) DAMv1/2 • Recombine UAS sim RNAi with UAS mCD8-GFP to label axons • Reduced sim? Fasciculation defects? BAmas1/2 sim 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 clones BAmas1/2 clones Trdm clones Axons in late sim mutant simH9/Df(3R)ry75 WT DAMv1/2 BAmas1/2? BAmas1/2 Trdm? Trdm sim BP106
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