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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.

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Presentation on theme: "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."— Presentation transcript:

1 Stephanie Freer Lab Meeting 4.13.10

2 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

3 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

4 Optic lobe expression sim GFP Laminar OL Neurons Medullar OL Neurons

5 Posterior central brain clusters Lateral Posterior Cluster sim GFP Small Posterior Cluster

6 Expression in the anterior clusters 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 DAMv1/2 BAmas1/2 Trdm

7 Further dissection of Intron 3 F2 1.4kb F1F3 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

8 Intron 3 F1-GAL4 x UAS mCD8-GFP AnteriorPosterior GFP expression - a subset of anterior central brain cells - medullar optic lobe expression (subset) - posterior clusters sim GFP DAMv1/2 BAmas1/2 DPM

9 Additional intron 3-GAL4 enhancer constructs Intron 3 Frag 3 Intron 3 F2-GAL4 – no expression Intron 3 F3-GAL4 – subset of medulla Generated 3 500bp overlapping constructs sim GFP F2 1.4kb F1F3 F1a F1b F1+ F2

10 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

11 Intron 3 F1b-GAL4 x UAS mCD8-GFP sim BP106 GFP  Trdm BAmas1/2 Intron 3 F1b GAL4 – anterior Trdm cluster - optic lobe medulla - staining weak in both cases

12 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?

13 Summary of Larval Expression DAMv1/2, BAmas1/2 Trdm Lateral posterior Small Posterior DPM Medulla OL Lamina OL MidlineOther Intron 3 GAL4xxx xx x Intron 3 F1 GAL4x x xx Intron 3 F2 GAL4 x Intron 3 F3 GAL4 x Intron 3 F1a GAL4 xxx xx Intron 3 F1b GAL4 x x Intron 3 F1+F2 GAL4 x xx 3.7 sim GAL4 x x 1.0kb GAL4xxxxxxxxx Frag 2 GAL4 x x

14 Intron 3-GAL4 enhancer constructs F2 1.4kb F1F3 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

15 Post-embryonic function of sim? 1. MARCM - sim mutants in posterior clusters 2. sim RNAi - axon fasciculation defects 3. Adhesive/Anti-adhesive proteins

16 DAMv1/2 sim + cell clusters send axons across the commissure and fasciculate together sim² WT Sim DAPI GFP Sim GFP MARCM (mosaic analysis with a repressible cell marker)  Generates homozygous marked/mutant cells from heterozygous precursors

17 Anterior MARCM GFP clones sim BP106 GFP   DAMv1/2

18 MARCM clones in the posterior sim clusters Posterior  Anterior sim BP106 GFP sim BP106 GFP  3 tightly fasciculated axonal bundles cross the commissure

19 Posterior mutant sim BP106 DCAD2 sim DCAD2    WT sim H9 /Df(3R)ry 75

20 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

21 WT and mutant DPM clones sim BP106 GFP  WT DPM clone sim H9 DPM clone

22 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 BAmas1/2 sim

23 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

24 Axons in late sim mutant DAMv1/2 Trdm BAmas1/2 sim BP106 Trdm? BAmas1/2? WT sim H9 /Df(3R)ry 75

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