A modest collection of midline crms
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A modest collection of Midline CRMs. Joe Pearson Lab Meeting 8/25/08. Does common expression indicate common regulation?. A. B. C. A. B. D. C. D. A. C. D. B. w. x. y. z. A. B. C. D. Goals. Clone minimal CRMs that drive midline primordium expression

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A modest collection of Midline CRMs

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A modest collection of midline crms

A modest collection of Midline CRMs

Joe Pearson Lab Meeting

8/25/08


Does common expression indicate common regulation

Does common expression indicate common regulation?

A

B

C

A

B

D

C

D

A

C

D

B

w

x

y

z

A

B

C

D


Goals

Goals

  • Clone minimal CRMs that drive midline primordium expression

  • Dissect and analyze midline primordium CRMs

  • Determine mechanisms governing common gene expression patterns


Midline primordium genes

Midline primordium genes

ab

argos

bib

bnb

cdi

cenB1A

CG13333

CG31145

CG8291

CG32594

CG3409

CG7224

ct

dve

CG9634

glec

hbs

HGTX

mfas

oc

vvl

sty

Tkr

sog

Sema-1b

btl

rho

rst

sim

Tl


A modest collection of midline crms

Midline Fragment Cloning Progress


Methods

Methods

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

  • Fluorescent antibody detection:

    • Rabbit a-GFP(a488)

    • Guinea Pig a-Sim(Cy3)

    • Mouse a-EN or Rat a-ELAV(a647)


Argos

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

argos 5’ Fragments

5’-2

5’-1

5’Fragments contain no midline CRMs.


Argos intron1 1

argos Intron1-1

GFP

SIM

St. 13: Sporadic PMG expression

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


Cg13333

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

CG13333 5’

5’

GFP

SIM

St. 13: AMG, PMG, and iVUMs

GFP

SIM


Cg13333 5 motifs

CG13333 5’ motifs

Dmel

Dsim

Dsec

Dyak

Dere

Dana

Dpse

Dper

Dwil

Dgri

Dmoj

Dvir

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAC

AGGTAG

AGGTAC

AGGTGG

AGGTGG

AGGTGG

AGGTGG

AGGTGG

AGGTGG

AGGTGG

AGGTGG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAG

AGGTAA

AGGTAG

CACGT

CACGT

CACGT

CACGT

CTCGT

CACGT

CACGT

CACGT

AGGTAG

AGGAAG

AGGAAG

PhyloGibbs identified AGGTRG as a repeated, conserved motif.


Cg13333 3

CG13333 3’

3’

  • 3’Fragment drives no midline expression.

  • Off-target integration?

  • Additional regions to test?


A modest collection of midline crms

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

glec Intron1

  • Intron1 contains no midline CRMs


Glec 5 2

glec 5’-2

GFP

SIM

St. 10: 2-3 cells express GFP

St. 10

GFP

SIM

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

GFP

SIM

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

EN

GFP

SIM

SIM

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


Glec 5 3

glec 5’-3

GFP

SIM

St. 11: MP1 (variable weak other cells)

ELAV

GFP

SIM

GFP

SIM

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

ELAV

GFP

SIM

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


Glec 3

glec 3’

GFP

SIM

St. 11: 2-3 cells per midline segment

EN

GFP

SIM

St. 13: AMG, weak PMG


A modest collection of midline crms

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


Mfas 5 frag intron1

mfas 5’frag/Intron1

GFP

SIM

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

EN

SIM

GFP

St. 12/13:VUMs, moving ventrally

EN

GFP

SIM

St. 16: VUMs


Conclusions lingering questions

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

Next Steps

  • More enhancer screening (10+12+20+10)

  • Identify “minimal” (1kb) midline CRMs (20)

  • Computationally analyze, test motif function

  • Compare co-expressed CRMs


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