s12

midline neurons and glia

CNS midline sim expression: early vs. late

s6 s8 s11

midline precursors

Late sim midline expression is restricted to glia, H-cell sib, iVUMs and MNB progeny

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?

zfh1 En zfh1/En/Sim VGlut/En/Sim

WT s13

Sim Lim1 En + simtaugfp

WT s17

sim contains two alternative promoters

CNS midline neurons and gliaCentral brain neuronsOptic lobes

Mesectodermal precursorsCNS midline primordium

Sim regulates axonogenesis in central brain neurons

sim²

WT

SimDAPI

D. Lau

Testing function of late Sim in midline development and transcription

•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

*

sim locus

Df(3R) ry75

simH9

del

ry75/simH9 embryos do not express Sim protein after mid-embryogenesis

ry75/simH9simH9/TM3 s16 s16

Simsim-Gal4; UAS-taugfp

Simsim-Gal4; UAS-taugfp

ry75/simH9 = simlate

Late Sim is not required for wrapper expression, but midline glia have mild positioning defects

wrapper + gfp

twi-GFP control s11 s11

s17 s17

s17 s16twi-GFP control

simlate

simlate simlate

simlate

Late Sim is not required for sim transcription

s13 s17sim sim

simlate simlate

Embryos lacking late sim have a midline axonogenesis defect

simH9/TM3

simlate

simlate

Sim+ CNS midline neurons are GABAergic or Glutamatergic

Gad1: GABAergic neuronal marker

VGlut: Glutamatergic neuronal marker

iVUMs/pMNB: En+, Sim+, Gad1+

H-cell sib: En-, Sim+, VGlut+

En Sim Gad1 En/Sim/Gad1 En/Sim/VGlut

Wt

Late sim does not regulate Lim1 or Gad1 expression in iVUMs/MNB progeny

simlate

Gad1 Lim1 Gad1/Lim1/simtaugfp

s15

Late sim regulates VGlut expression in H-cell sib

CG13565 CG13565

s16 s16

70% 30%VGlut/simtau VGlut/simtau

simlate simlate

simlate simlate

en mutants have ectopic CNS midline VGlut expression

Sim VGlut Gad1

Wt

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

H-sib axon H-sib axon

Wt

Per-Gal4, UAS-tau::gfp; UAS-engrailed

CG13565/En/PerGad1/VGlut/Per

Proposed mechanism for midline VGlut transcriptional regulation

VGlutmVUM CRMH-sib CRM

1) H-cell sib and mVUMs exhibit different levels of VGlut expression2) H-sib VGlut expression can be repressed by en3) H-sib VGlut expression requires late Sim

Sim

RNA pol II

glutamatergic H-cell sib

Proposed mechanism for midline VGlut transcriptional regulation

VGlutmVUM CRMH-sib CRM

Sim

RNA pol II

glutamatergic H-cell sib

1) Late Sim is co-expressed in H-cell sib with at least 4 other TFs (Per, Lim1, Fkh, Nvy)

X

Proposed mechanism for midline VGlut transcriptional regulation

VGlutmVUM CRMH-sib CRM

Sim

RNA pol II

GABAergiciVUM/pMNB

1) En mutants exhibit ectopic VGlut expression in Sim+ neurons2) En misexpression in H-cell sib represses VGlut

En

Proposed mechanism for midline VGlut transcriptional regulation

VGlutmVUM CRMH-sib CRM

Sim

RNA pol II

This model presumes En and Sim directly regulates VGlut

Testable via identification of midline CRMs and En/Sim binding sites

En

GABAergiciVUM/pMNB

VGlut genomic locus

14.5 kb

2.5 kb

En

En

En En EnEn

10 kb

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

Sim Sim

Sim Sim

Transgenic expression analysis

En

En

En En EnEn

Sim Sim

Sim Sim

Transgenic expression analysis

2 independent insertions

No embryonic expression

En

En

En En EnEn

Sim Sim

Sim Sim

Transgenic expression analysis

CNS: 2 cells/ hemisegment in thoracic segments 1 cell/hemisegment in abdominal segments

Outside CNS during mid-embryogenesis

Only 1 insertion line

En

En

En En EnEn

Sim Sim

Sim Sim

Transgenic expression analysis

CNS: 2 cells/ hemisegment in thoracic segments 1 cell/hemisegment in abdominal segments

Outside CNS during mid-embryogenesis

Only 1 insertion line

En

En

En En EnEn

Sim Sim

Sim Sim

Transgenic expression analysis

2 independent insertions

Expression not determined

En

En

En En EnEn

Sim Sim

Sim Sim

Transgenic expression analysis

TA cloned

Not subcloned into stinger

En

En

En En EnEn

Sim Sim

Sim Sim

Conclusions Future Directions

• ry75/simH9 allelic combination eliminates late sim expression in midline glia and iVUMs, H-cell 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)

• simlate mutants have normal midline Gad1 expression, but fail to express VGlut in H-cell sib (~70% segments)

• En misexpression in H-cell sib phenocopies simlate H-cell sib phenotype

• En represses VGlut in GABAergic midline neurons, preventing activation by a TF complex containing Sim.

• 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)

• 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

• Test other H-cell sib TF mutants for VGlut expression