Transcript CNS midline sim expression: early vs. late midline precursors s6 s8 midline neurons and glia s12 s11 Late sim midline expression is restricted to glia, H-cell sib, iVUMs and.

CNS midline sim expression: early vs. late
midline
precursors
s6
s8
midline
neurons and glia
s12
s11
Late sim midline expression is restricted to
glia, H-cell sib, iVUMs and MNB progeny
WT
s13
zfh1
WT
En
zfh1/En/Sim
Lim1
En + simtaugfp
VGlut/En/Sim
s17
Sim
What is the function of late Sim in mature midline neurons and glia?
What are late Sim direct target genes?
Does Sim interact with other TFs to activate late gene expression? If so,which ones?
sim contains two alternative promoters
CNS midline neurons and glia
Central brain neurons
Optic lobes
Mesectodermal precursors
CNS midline primordium
Sim regulates axonogenesis in central brain
neurons
WT
Sim
DAPI
sim²
D. Lau
Testing function of late Sim in midline
development and transcription
sim locus
Df(3R) ry75
del
simH9
*
•Df(3R) ry75 removes Sim late promoter leaving early sim expression intact
•simH9 is a null point mutation that introduces an early stop codon in the protein
•simH9/TM3 twi-GFP X Df(3R) ry75/TM3 twi-GFP
--assay GFP-negative embryos using AP ISH
•simH9/TM3 twi-GFP X w; {3.7sim-gal4,w+}, {UAS-tau::gfp,w+}; Df(3R) ry75/TM3 ftz-lacZ
--assay twi-GFP- and lacZ-negative embryos using FISH/antibody staining
--3.7sim-Gal4<UAS-tau::gfp marks all midline cells
ry75/simH9 embryos do not express
Sim protein after mid-embryogenesis
simH9/TM3
Sim
sim-Gal4; UAS-taugfp
s16
ry75/simH9
Sim
sim-Gal4; UAS-taugfp
ry75/simH9 = simlate
s16
Late Sim is not required for wrapper expression,
but midline glia have mild positioning defects
twi-GFP control
s11
simlate
s11
wrapper + gfp
simlate
s17
simlate
s17
twi-GFP control
s17
simlate
s16
Late Sim is not required for sim transcription
simlate
simlate
s13
sim
s17
sim
Embryos lacking late sim have a midline
axonogenesis defect
simlate
simH9/TM3
simlate
Sim+ CNS midline neurons are
GABAergic or Glutamatergic
Wt
En
Sim
Gad1
En/Sim/Gad1
Gad1: GABAergic neuronal marker
VGlut: Glutamatergic neuronal marker
iVUMs/pMNB: En+, Sim+, Gad1+
H-cell sib: En-, Sim+, VGlut+
En/Sim/VGlut
Late sim does not regulate Lim1 or Gad1
expression in iVUMs/MNB progeny
simlate
s15
Gad1
Lim1
Gad1/Lim1/simtaugfp
Late sim regulates VGlut expression in
H-cell sib
simlate
simlate
VGlut/simtau
simlate
70%
s16
CG13565
VGlut/simtau
simlate
30%
s16
CG13565
en mutants have ectopic CNS midline
VGlut expression
Wt
Sim
VGlut
Gad1
en
Sim
VGlut
Tbh
en mutants: 2-4 VGlut+ Sim+ neurons/segment
Misexpression of En in H-cell sib represses
VGlut and does not activate Gad1
En
VGlut
PerGal4<tau
Wt
Gad1/VGlut/Per
CG13565/En/Per
H-sib axon
Per-Gal4, UAS-tau::gfp; UAS-engrailed
H-sib axon
Proposed mechanism for midline
VGlut transcriptional regulation
glutamatergic
H-cell sib
RNA pol II
Sim
VGlut
H-sib CRM
1)
2)
3)
mVUM CRM
H-cell sib and mVUMs exhibit different levels of VGlut expression
H-sib VGlut expression can be repressed by en
H-sib VGlut expression requires late Sim
Proposed mechanism for midline
VGlut transcriptional regulation
glutamatergic
H-cell sib
RNA pol II
Sim
X
VGlut
H-sib CRM
1)
mVUM CRM
Late Sim is co-expressed in H-cell sib with at least 4 other TFs (Per, Lim1, Fkh, Nvy)
Proposed mechanism for midline
VGlut transcriptional regulation
GABAergic
iVUM/pMNB
RNA pol II
Sim
En
VGlut
H-sib CRM
1)
2)
mVUM CRM
En mutants exhibit ectopic VGlut expression in Sim+ neurons
En misexpression in H-cell sib represses VGlut
Proposed mechanism for midline
VGlut transcriptional regulation
GABAergic
iVUM/pMNB
RNA pol II
Sim
En
VGlut
H-sib CRM
mVUM CRM
This model presumes En and Sim directly regulates VGlut
Testable via identification of midline CRMs and En/Sim binding sites
VGlut genomic locus
14.5 kb
10 kb
Sim Sim
En
Sim
En
En
En
2.5 kb
En
En Sim
6 putative En binding sites (YAATYANB) identical from melanogaster to virilis
3 putative Sim binding sites (ACGTG) identical from melanogaster to virilis
Genomic fragments cloned into GFP expression P-element for injection
Transgenic expression analysis
Sim
En
Sim
En
En
Sim
En
En
En Sim
Transgenic expression analysis
Sim
En
Sim
En
En
Sim
En
En
En Sim
2 independent insertions
No embryonic expression
Transgenic expression analysis
Sim
En
Sim
En
En
Sim
En
En
En Sim
CNS: 2 cells/ hemisegment in thoracic segments
1 cell/hemisegment in abdominal segments
Outside CNS during mid-embryogenesis
Only 1 insertion line
Transgenic expression analysis
Sim
En
Sim
En
En
Sim
En
En
En Sim
CNS: 2 cells/ hemisegment in thoracic segments
1 cell/hemisegment in abdominal segments
Outside CNS during mid-embryogenesis
Only 1 insertion line
Transgenic expression analysis
Sim
En
Sim
En
En
Sim
En
En
En Sim
2 independent insertions
Expression not determined
Transgenic expression analysis
Sim
En
Sim
En
En
Sim
En
En
En Sim
TA cloned
Not subcloned into stinger
Conclusions
•
ry75/simH9 allelic combination eliminates late
sim expression in midline glia and iVUMs, Hcell sib, and pMNB
•
simlate mutants express wrapper, but may
have a MG defect.
•
simlate mutants exhibit ectopic midline axonal
projections to the segmental nerve root
(likely iVUM axons)
•
•
•
Future Directions
•
Manipulate late Sim function using
misexpression of dominant-negative sim
constructs; Per-Gal4 mediated expression
could test neuron specific Sim function
•
en; simlate double mutant to test requirement
of sim on formation of ectopic glutamatergic
Sim+ neurons ( or use expression of sim
dominant-negative constructs in en mutant)
•
simlate mutants have normal midline Gad1
expression, but fail to express VGlut in Hcell sib (~70% segments)
Identify H-cell sib CRM in VGlut locus and
test dependence on Sim and En
•
Test panel of neuronal/glial markers to
identify additional late Sim targets
En misexpression in H-cell sib phenocopies
simlate H-cell sib phenotype
•
Test other H-cell sib TF mutants for VGlut
expression
En represses VGlut in GABAergic midline
neurons, preventing activation by a TF
complex containing Sim.