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