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