a modest collection of midline crms joe pearson lab meeting 8/25/08

A modest collection of Midline CRMs

Joe Pearson Lab Meeting8/25/08

Does common expression indicate common regulation?

A

B

C

DC D

A B

A

B C D

C DA B

w x y z

Goals

• Clone minimal CRMs that drive midline primordium expression

• Dissect and analyze midline primordium CRMs• Determine mechanisms governing common

gene expression patterns

CG13333

Midline primordium genesargosab bib bnb

btl

cdi cenB1A CG31145

CG32594 CG3409 CG7224 CG8291

ct dve glec

hbs HGTX mfas oc

rhoSema-1b

sog sty vvlTkr

CG9634

rst sim Tl

Gene # fragments(transformed)

Gene # fragments(transformed)

argos 5(4) CG7224 1(0)

bib 3(2) CG8291 1(1)

bnb 4(0) CG9634 2(0)

cdi 2(0) glec 5(4)

cenb1a 2(1) HGTX 8(2)

CG13333 2(2) mfas 3(3)

CG32594 4(3) Sema-1b 1*

CG3409 4(0) sty 6(4)

Midline Fragment Cloning Progress

Methods

• Lines tested: Fragments with potential midline expression (live GFP, GFP in situ, or -GFP histochemical)

• Fluorescent antibody detection:– Rabbit -GFP(a488)– Guinea Pig -Sim(Cy3)– Mouse -EN or Rat -ELAV(a647)

argos

Expression•St. 11 midline expression (subset)•St. 13+ midline glial expression (strong AMG)

Locus•37 kb noncoding locus (18.5 kb tested)•5 Fragments cover 5’ + Introns•4 transformed, 3 tested

argos 5’ Fragments

5’-1 5’-2

5’Fragments contain no midline CRMs.

argos Intron1-1

St. 13: Sporadic PMG expressionSt. 14+: Expression in 1-2 AMG, PMG fade into the ether

GFP SIM

CG13333

Expression•St. 11 midline expression (all)•St. 13+ midline glial expression•St. 15 midline expression weakens

Locus•1.5 kb noncoding locus•2 Fragments cover 5’ + 3’•2 transformed, 2 tested

CG13333 5’

5’

St. 13: AMG, PMG, and iVUMs

GFP SIM

GFP SIM

CG13333 5’ motifs

AGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTACAGGTAGAGGTAC

DmelDsimDsecDyakDereDanaDpseDperDwilDgriDmojDvir

AGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTAG

AGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAAAGGTAG

CACGTCACGTCACGTCACGTCTCGTCACGTCACGTCACGT

AGGTAGAGGAAGAGGAAG

PhyloGibbs identified AGGTRG as a repeated, conserved motif.

CG13333 3’

3’

•3’Fragment drives no midline expression.•Off-target integration?•Additional regions to test?

glec

Expression•St. 8 midline expression (all)•refines to 6-7 cells by St. 11•St. 13+ midline glial expression

Locus•16.5 kb noncoding locus•5 Fragments cover 5’, Intron1, 3’•4 transformed, 4 tested

glec Intron1

•Intron1 contains no midline CRMs

glec 5’-2

St. 10

St. 10: 2-3 cells express GFP

St. 11: 2-3 cells (AMG?) express strongly, others may be expressing weakly

St. 13: AMG, MP1, H-cell/Sib, and variable VUMs

St. 16: MG, VUMs, MNB?, H-Cell/Sib, MP1

GFP SIM

GFP SIM

GFP SIM

GFP SIM SIMEN

glec 5’-3

St. 11: MP1 (variable weak other cells)

St. 13/14: MP1, variable VUMs, AMG, H-Cell sib

St. 16: MP1, weak H-Cell/Sib, some AMG

GFP SIM

GFP SIM GFP SIM

GFP SIMELAV

ELAV

glec 3’

St. 11: 2-3 cells per midline segment

St. 13: AMG, weak PMG

GFP SIM

GFP SIMEN

mfas

Expression•St. 10 midline expression (all)St. 12+ midline glia

Locus•7.2 kb noncoding locus•3 Fragments cover 5’ & Intron1•3 transformed, 2 tested

St. 12/13:VUMs, moving ventrally

St. 16: VUMs

mfas 5’frag/Intron1

St. 11: 4 basal, and 2 apical cells (what are they?)GFP SIM

GFP SIM

GFP SIMEN

EN

Conclusions/Lingering Questions

• “Midline primordium” expression may be amalgam of multiple CRMs

• Multiple regulatory modes involved• Can these elements be “cleaned up”?• Have I captured full elements?

Next Steps

• More enhancer screening (10+12+20+10)• Identify “minimal” (1kb) midline CRMs (20)• Computationally analyze, test motif function• Compare co-expressed CRMs