Download - Stephanie Freer Lab Meeting 7.6.09
Stephanie Freer
Lab Meeting 7.6.09
Post-embryonic expression of sim
• 3 anterior Sim+ clusters• 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 tau-GFP 2. Analyze embryonic and larval brains for GFP-expression in sim+ cells3. Further dissect fragments not restricted to central brain clusters
Construct Embryonic Expression Larval Expression
1.0 kb all midline cells none2.8 kb midline glia, muscle precursors small posterior clusters3.1 kb hindgut none5.5 kb foregut, pharynx none3.7 kb all midline cells ventral anterior clusters (FLP out)sandman midline glia, muscle precursors small posterior clusters
Frag 1 coming soon -Frag 2 midline glia, muscle precursors laminar cells (OL), small post.
clustersFrag 3 CNS central brain NBs, GMCsFrag 4 none noneFrag 5 gut noneIntron 3 CNS medullar cells (OL), ant./post.
clustersIntron 7 CNS, PNS CNS3’sim F1 none none3’sim F2 - -3’sim F3 CNS anterior lateral clusters (timeout?)3’sim F4 none none
sim transgenic lines
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
Additional intronic enhancers
Intron 3 Frag 2 Anterior
3 Intron 3 500bp fragments
Intron 3 Frag 1 – posterior clusters
- subset of anterior clusters?
- optic lobes
Intron 3 Frag 2 – no expression
Intron 3 Frag 3 – Optic lobes (medullar subset)
New fragment spanning intron 4-6 to be injected
Intron 3 Frag 3
Intron 3 F1-GAL4 x UAS mCD8-GFP
Anterior Posterior
nc82GFP
GFP expression - a subset of anterior central brain cells - medullar optic lobe expression (subset) - posterior clusters
Reduction of late sim leads to structural defects in the adult
• simJ1-47 - temperature sensitive allele• Neuropil disorganization in the adult• Sterile with behavioral defects
WT simJ1-47
Rein et al., 2002
Neckameyer and Matsuo, 2008
Central complex structure
Fan-shaped body – visual pattern learning Ellipsoid body, noduli (ebo) – decay in walking activityProtocerebral bridge (nob) – deficits in step length
Abnormal turning behavior, “left-right bargaining” in mutants of both eb and pb defects typically at the midline
Intron 3-GAL4 expression in the adult brain
nc82GFP
Expression in a subset of fan-shaped body axons
Lack of expression in the protocerebral bridge and ellipsoid body
In general, lots of axons stained specificity?
Intron 3-GAL4 expression in the optic lobe
nc82GFP
Expression in the SOG
nc82GFP
Intron 3 F1-GAL4 x UAS mCD8-GFP
Expression in the anterior subeosphogeal ganglion, send projections dorsallyCorresponds to Trdm?
Summary of adult expression
nc82GFP
Intron 3 F1-GAL4 x UAS mCD8-GFP
Expression in the SOG, optic lobes, PNs in the AL, fan-shaped body, MBs, superior medial protocerebrum, superior lateral protocerebrum
Further dissection of Intron 3
1.4kb
F1
F2
F3
F1a
F1b
F1+ F2
1. Separate optic lobe expression from central brain expression2. Capture all central brain neurons within one line
Another possibility: Mutate potential optic lobe elements
Transmitter phenotype of sim-expressing midline cells in the embryo
Sim
En/Sim
VGlut/Sim
Gad1/Sim
* **
Posterior VGlut expression
VGlutSim
VGlut RNA co-localizes with Sim protein
Neurotransmitter phenotype in posterior sim-positive neurons VGlut
VGlut Fragment 4-6 in the 3rd instar brain
SimGFP
EnEn
En En EnEn
Sim SimSimSim
CG3557
2.4 kb
2.5 kb2.8 kb
3.7 kb2.7 kb
4.8 kb
0.8 kb 0.6 kb
Frag 2Frag 8
Frag 4
Frag 1
Frag 6
Frag 5Frag 9Frag 7
intr
on
1
intron 3
intr
on
2
VGlut Antibody in 3rd Instar Larvae
VGlutTau
No apparent protein co-localization at the cell bodies VGlut protein in transit to the synapse
VGlut staining absent in secondary lineages labeled by BP106 (Daniels et al. 2008)
Transmitter phenotype in anterior sim-positive central brain clusters
• 1-2 VGlut+, sim+ cells observed for each cluster
• No Gad1+, sim+ cells observed
Two options:
1. Cells not differentiated yet Stain pupal and adult brains
2. Another transmitter involved- monoamines: dopamine, serotonin, octopamine- neuropeptides: neuropeptide F
SimGad1VGlut
Medial Cluster
Summary
Post-embryonic expression within Intron 3
Expression from intron 3-GAL4 in the adult
- Subset in the fan-shaped body
- Structures other than the CC
Posterior clusters are glutamatergic
• Analyze additional fragments
- Frag 1, other introns, 3’ sim F2
- Intron 3 dissection• Neurotransmitter staining in pupae and
adults• Behavioral Analysis
- Larval and adult locomotion