lab meeting #1

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