Post on 11-Jan-2016
description
transcript
Lab Meeting #1
9-13-2010
Expression profiles and transcriptional networks in the
CNS midline
Wheeler et al., 2006
High-throughput sequencing of the transcriptome
Sequenceall fragments
Isolate RNA Fragment RNAGenerate adapter-ligated cDNA
Steps to RNA-seq
Isolate Cells Purify RNA Prepare cDNA Sequence Analyze
Find midline specific GAL4 driver
Stage embryos @ 25oC
Dissociate cells
Sort cells via FACS
Quest for a midline specific driver
Strong GFP expression in midline
No (or very little) GFP expression outside of midline
Expressed early, stays on late
3.7sim-GAL4 > UAS-mCD8-GFP (LL5)
1.0sim-GAL4 > UAS-mCD8-GFP (LL5)
sema1b-I1F3sC-GAL4 > UAS-mCD8-GFP (LL5)
3.7sim-GAL4 > UAS-tau-GFP on Chromosome II
3.7sim > LL5
3.7sim > LL5
3.7sim > LL5
3.7sim > LL5
1.0sim > LL5
sema1b > LL5
3.7sim > tauGFP on II
Unforeseen problem with 3.7sim > tau-GFP on II
Embryos @ 25oC for 14 -16 hours : all should be stage 16 – 17
Actual: 56% Stage 16+14% Stage 13-169% Stage 11-1221% Dead
N=132
Possible quick fix : 3.7sim > tau-GFP on IIIProblem: Flies do not lay very well on plates
Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo.
Phosphorylation differentiates tau-dependent neuronal toxicity and dysfunction.
Differential effects of Tau on the integrity and function of neurons essential for learning in Drosophila.
Tau and tau reporters disrupt central projections of sensory neurons in
Drosophila.
Alternatives to tau-GFP
3.7sim-GAL4
UAS-mCD8-GFP (LL4) : on X
UAS-mCD8-GFP (LL5) : on II
UAS-mCD8-GFP (LL6) : on III
Ectopic expression outside midline
Specific, but weak expression
Strong and specific
3.7sim > LL6
3.7sim > LL6
3.7sim-GAL4/CyO ; UAS-mCD8-GFP-LL6
17-21 hrs @ 25oC: all should be stage 17 or later
2% Stage 13-1484% Stage 17+14% Dead N = 120
3.7sim-GAL4 ; UAS-mCD8-GFP-LL6
16-18 hrs @ 25oC: all should be stage 17+
97% Stage 17+3% Dead N = 91
Cell Dissociation
Cell Dissociation
With 3.7sim > tau-GFP
2 hr collection ~75 – 100 uL of embryos
~20,000 cells / uL or ~20 x 106 cells / collection
Fluorescence Activated Cell Sorting(FACS)
w1118 3.7sim > tau-GFP presort
Presort with PI Sorted into FACS buffer
Sorted into PBS FACS buffer
Current Status
Isolate Cells Purify RNA Prepare cDNA Sequence Analyze
Find midline specific driver
Stage embryos
Dissociate cells
Sort cells via FACS
Future Direction3.7sim-QF > QUAS-mtdTomato-3xHA
CoolEnhancer-GAL4 > UAS-GFP
CG32105
Otx
(orthodenticle,ocelliless)
Lmx1a Msx1 Nkx6.1(HGTX)
(indirectly)
Ngn2,(tap)
TH,(pale)
Nurr1,(Hr38)
Pitx3(Ptx1)
* **
*1 1
RuntCG321053.7sim > tauGFP
T1 T2
11
**
**
RuntCG321053.7sim > tauGFP
Abdominal segments
A1 A2T2 T3RuntCG32105
*
T2
1
T3
*1
A1 A2
* *
*1 11
A1 A2 A1 A2RuntCG32105
A1 A2
* *
11
A1 A2
* *
*1 11
A3 A4 A5RuntCG32105
**
A3
1
* **
A4
1
* *(*)
A5
1
Stage 11
** *
* **
***
Stage 12-anterior segments
zfh1CG32105
A7 A8
**
*
A7 A8
* ** **
A7 A8
*
zfh1CG32105
*
Summary of CG32105 expression
Stage 11 – not expressed
Stage 12 – expressed in mVUMs of all segments looked at mVUM4 and 5 early mVUM4, 5, and 6 later
Stage 15-16 – not expressed in thoracic mVUMs may be expressed in 1-2 thoracic MNBp expressed in abdominal mVUMs
-maybe not all mVUMs in anterior abdominal segments
will die slowly (wds)
w1118
w1118
w1118
elav-GAL4 > UAS-wds