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 MP1 PMG RuntEn

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-runtUAS-en Wildtype UAS-runtUAS-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 Runt EnRunt

Consequences of converting AMG to PMG or PMG to AMG wildtype UAS-runtUAS-en late 16 GFP wrapperwrapper GFP wrapper GFP

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L(1)sc l(1)sc en runt?

L(1)sc Stg odd y 1 w 1 f 1 run 3 12/1 L(1)sc Stg runt negatively regulates l(1)sc Does engrailed regulate l(1)sc? L(1)sc Stg UAS-runt UAS-enenE/enE wildtype

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 8 th (end of March for UNC?)

AMG MP1 PMG RuntEn

AMG MP1 PMG runt hblim3HGTXpdfodd

AMG MP1 PMG hb ? ? ? ? Odd, HGTX, pdf, lim3?

sim::Gal4 driving uas-hb generates extra lim-3 HGTX + cells WT control (no HGTX staining) UAS-hb stg, uas-hb WT Lim3 GFP HGTXLim3 HGTX

Wrapper and lim3 co-localize in stg uas-hb embryos Lim-3 + wrapper + neurons Lim-3 + wrapper - neuron lim3wrapperstg

Stop codon deletion

Lim3 and odd are both expressed in hb12 alleles lim3 odd stg

AMG MP1 PMG runt hblim3HGTXpdfodd

Ectopic Runt expression does not affect Lim3 expression GFP En Lim3 WT stage 13 Stg uas-runt

stg, uas-runt stg, uas-hb WT

Driving engrailed in the midline reduces odd expression GFP odd Runt

runt mutants lack odd staining GFP odd 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 siteHb binding site Runt binding site 1 kb

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

eve enftz wingless slp enwingless gooseberry-neuro – MP3s patched

Establishing early midline identity A, B l(1)sc en, PMG C runt MP1, hb, odd D, E runt, slp1 and 2 AMG F slp1 and 2, gsb-n, l(1)sc MP3 H en MNB, Vums G eve, 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 siteRunt 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