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A modest collection of Midline CRMs
Joe Pearson Lab Meeting8/25/08
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Does common expression indicate common regulation?
A
B
C
DC D
A B
A
B C D
C DA B
w x y z
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Goals
• Clone minimal CRMs that drive midline primordium expression
• Dissect and analyze midline primordium CRMs• Determine mechanisms governing common
gene expression patterns
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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
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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
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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)
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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
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argos 5’ Fragments
5’-1 5’-2
5’Fragments contain no midline CRMs.
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argos Intron1-1
St. 13: Sporadic PMG expressionSt. 14+: Expression in 1-2 AMG, PMG fade into the ether
GFP SIM
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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
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CG13333 5’
5’
St. 13: AMG, PMG, and iVUMs
GFP SIM
GFP SIM
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CG13333 5’ motifs
AGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTACAGGTAGAGGTAC
DmelDsimDsecDyakDereDanaDpseDperDwilDgriDmojDvir
AGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTGGAGGTAG
AGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAGAGGTAAAGGTAG
CACGTCACGTCACGTCACGTCTCGTCACGTCACGTCACGT
AGGTAGAGGAAGAGGAAG
PhyloGibbs identified AGGTRG as a repeated, conserved motif.
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CG13333 3’
3’
•3’Fragment drives no midline expression.•Off-target integration?•Additional regions to test?
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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
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glec Intron1
•Intron1 contains no midline CRMs
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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
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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
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glec 3’
St. 11: 2-3 cells per midline segment
St. 13: AMG, weak PMG
GFP SIM
GFP SIMEN
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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
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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
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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?
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Next Steps
• More enhancer screening (10+12+20+10)• Identify “minimal” (1kb) midline CRMs (20)• Computationally analyze, test motif function• Compare co-expressed CRMs