Stephanie Freer Lab Meeting

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 + 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 Lateral Posterior Cluster sim GFP Small Posterior Cluster

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

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

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

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

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 sim BP106 GFP  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 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 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

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

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

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

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

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 sim BP106 GFP  WT DPM clone sim H9 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 BAmas1/2 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 DAMv1/2 Trdm BAmas1/2 sim BP106 Trdm? BAmas1/2? WT sim H9 /Df(3R)ry 75