1/25/10

runt and engrailed-dependent specification of midline glia

Outline

runt and engrailed specify AMG and PMG, respectively

The role of hunchback and runt in the specification of MP1

Establishing early midline fate

Runt and the midline

Downstream targets of runt

Key developmental time points – AMG and PMG

AMG

AMG

AMG

AMG

AMG

AMG MP1

MP1

PMG PMG

PMG

PMG

Runt En

Distinct expression patterns in AMG vs PMG

Runt and Engrailed are spatially restricted to either AMG or PMG

runt and engrailed control AMG and PMG gene expression, respectively

Wildtype UAS-runt UAS-en Wildtype UAS-runt UAS-en

Previous work

Klinger and Gergen, 1993

runt expression

WT

hs-Runt

engrailed expression

Aronson, et al, 1997

wildtype

Runt and Engrailed cross-repress each other in order to generate AMG and PMG

When are expression domains established?

Runt En GFP Wrapper

13 sag 13 sag 13 sag 13 sag

Runt En Runt

Consequences of converting AMG to PMG or PMG to AMG

wildtype

UAS-runt UAS-en

late 16

late 16 late 16

GFP wrapper wrapper

GFP wrapperGFP

?

L(1)sc

l(1)sc en

runt?

L(1)sc Stg odd

y1 w1 f1 run3

12/1L(1)sc Stg

L(1)sc Stg

runt negatively regulates l(1)sc

Does engrailed regulate l(1)sc?

L(1)sc Stg

UAS-runt

UAS-en enE/enE

wildtype

L(1)sc

L(1)sc en

runt

?

Remaining experiments

•Finish images for runt and engrailed mutant backgrounds

•Quantify glial cells in UAS-runt and UAS-en backgrounds

•Determine role of L(1)sc

•Determine the spatial and temporal expression of Runt and Engrailed

1. Mechanisms used to generate MP1s

2. Establishment of early midline fate

3. Transcriptional regulation of AMG gene expression

The specification of neuron and glial cell fate by the transcription factor Runt

Due April 8th (end of March for UNC?)

AMG

AMG

AMG

AMG

AMG

AMG MP1

MP1

PMG PMG

PMG

PMG

Runt En

AMG

AMG

AMG

AMG

AMG

AMG MP1

MP1

PMG PMG

PMG

PMG

runt hb lim3 HGTX pdf odd

AMG

AMG MP1

MP1

PMG PMG

PMG

PMG

hb

?

?

?

?

Odd, HGTX, pdf, lim3?

sim::Gal4 driving uas-hb generates extra lim-3 HGTX + cells

WT control (no HGTX staining)

UAS-hb

stage 11 stage 13 stage 15 stage 16 stage 17lim3 0.2 3 3.423077 3.043478 3.210526

stg, uas-hb

WT

wt stage 11 stage 13 stage 15 stage 16 stage 17lim3 0 2 2 2 2

Lim3 GFP HGTX Lim3 HGTX

Wrapper and lim3 co-localize in stg uas-hb embryos

Lim-3 + wrapper + neurons

Lim-3 + wrapper - neuron

lim3 wrapperstg

Stop codon

deletion

Lim3 and odd are both expressed in hb12 alleles

lim3

odd

stg

AMG

AMG

AMG

AMG

AMG

AMG MP1

MP1

PMG PMG

PMG

PMG

runt hb lim3 HGTX pdf odd

Ectopic Runt expression does not affect Lim3 expression

GFP En Lim3 GFP En Lim3

WT stage 13 Stg uas-runt

stage 11 stage 13 stage 15 stage 16 stage 17lim3 0.00 2.04 2.09 2.00 2.00

stg, uas-runt

stage 11 stage 13 stage 15 stage 16 stage 17lim3 0.2 3 3.423077 3.043478 3.210526

stg, uas-hb

WT

wt stage 11 stage 13 stage 15 stage 16 stage 17lim3 0 2 2 2 2

Driving engrailed in the midline reduces odd expression

GFP odd RuntGFP odd Runt

runt mutants lack odd staining

GFP odd GFP odd

12/0 12/0

Hunchback and Runt?

HB

odd HGTX lim3 pdf

Runt

MP1 transcription factors

Lim3 ectopic expression?

Uas-odd

Uas-ftz

Uas-exex

X

X

X

exex-Lim3 normal

Aim 1 Methods and goals:

• Test available mutants and uas lines for altered MP1 fate• Morphology• gene expression

• Identify MP1 enhancer• Hunchback, Runt, and Engrailed binding sites• Mutate sites and identify

• Microarray MP1 neurons• Isolate MP1s using glec enhancer and sim-GAL4 UAS-TAU-GFP (2-color sorting)• Amplify and hybridize to affy microarray chip or use chip seq

Sim binding site Hb binding siteRunt binding site

1 kb

Aim 2 – Early specification of midline by runt and additional transcription factors

eve

enftzwingless

slp

enwingless

wingless

gooseberry-neuro – MP3spatched

Establishing early midline identity

A, Bl(1)sc

en, PMG

Crunt

MP1, hb, odd

D, Erunt, slp1 and 2

AMG

Fslp1 and 2, gsb-n, l(1)sc

MP3

Hen

MNB, Vums

Geve, en

• Test available mutants and uas lines for altered midline• Morphology• Gene expression

Aim 2: Establishing early midline identity

•Based on results, determine which transcription factors specify each cell type

Aim 3: Identify the transcriptional mechanisms used to generate AMG and PMG

Goals:

Identify Runt binding sites within AMG cis-regulatory regions

Identify Engrailed binding sites

Determine if Engrailed and Runt function in conjunction with Sim to specify AMG and PMG cell fate

Sim binding site Runt binding site

1 kb

CG8776

Summary

runt and engrailed co-repress each other to specify AMG and PMG fates, respectively

runt is necessary to generate MP1s, hunchback is sufficent

Further clues pertaining to midline development should arise from uas lines and genetic analysis