CNS midline sim expression early vs late midline

CNS midline sim expression: early vs. late midline precursors s 6 s 8 midline neurons and glia s 12 s 11

Late sim midline expression is restricted to glia, H-cell sib, i. VUMs and MNB progeny WT s 13 zfh 1 WT En zfh 1/En/Sim Lim 1 En + simtaugfp VGlut/En/Sim s 17 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(3 R) ry 75 del sim. H 9 * • Df(3 R) ry 75 removes Sim late promoter leaving early sim expression intact • sim. H 9 is a null point mutation that introduces an early stop codon in the protein • sim. H 9/TM 3 twi-GFP X Df(3 R) ry 75/TM 3 twi-GFP --assay GFP-negative embryos using AP ISH • sim. H 9/TM 3 twi-GFP X w; {3. 7 sim-gal 4, w+}, {UAS-tau: : gfp, w+}; Df(3 R) ry 75/TM 3 ftz-lac. Z --assay twi-GFP- and lac. Z-negative embryos using FISH/antibody staining --3. 7 sim-Gal 4<UAS-tau: : gfp marks all midline cells

ry 75/sim. H 9 embryos do not express Sim protein after mid-embryogenesis sim. H 9/TM 3 Sim sim-Gal 4; UAS-taugfp s 16 ry 75/sim. H 9 Sim sim-Gal 4; UAS-taugfp ry 75/sim. H 9 = simlate s 16

Late Sim is not required for wrapper expression, but midline glia have mild positioning defects twi-GFP control s 11 simlate s 11 wrapper + gfp simlate s 17 twi-GFP control s 17 simlate s 16

Late Sim is not required for sim transcription simlate s 13 sim s 17 sim

Embryos lacking late sim have a midline axonogenesis defect simlate sim. H 9/TM 3 simlate

Sim+ CNS midline neurons are GABAergic or Glutamatergic Wt En Sim Gad 1 En/Sim/Gad 1: GABAergic neuronal marker VGlut: Glutamatergic neuronal marker i. VUMs/p. MNB: En+, Sim+, Gad 1+ H-cell sib: En-, Sim+, VGlut+ En/Sim/VGlut

Late sim does not regulate Lim 1 or Gad 1 expression in i. VUMs/MNB progeny simlate s 15 Gad 1 Lim 1 Gad 1/Lim 1/simtaugfp

Late sim regulates VGlut expression in H-cell sib simlate VGlut/simtau simlate 70% s 16 CG 13565 VGlut/simtau simlate 30% s 16 CG 13565

en mutants have ectopic CNS midline VGlut expression Wt Sim VGlut Gad 1 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 Gad 1 En VGlut Per. Gal 4<tau Wt Gad 1/VGlut/Per CG 13565/En/Per H-sib axon Per-Gal 4, UAS-tau: : gfp; UAS-engrailed H-sib axon

Proposed mechanism for midline VGlut transcriptional regulation glutamatergic H-cell sib VGlut RNA pol II Sim H-sib CRM m. VUM CRM 1) H-cell sib and m. VUMs exhibit different levels of VGlut expression 2) H-sib VGlut expression can be repressed by en 3) H-sib VGlut expression requires late Sim

Proposed mechanism for midline VGlut transcriptional regulation glutamatergic H-cell sib VGlut RNA pol II Sim X H-sib CRM m. VUM CRM 1) Late Sim is co-expressed in H-cell sib with at least 4 other TFs (Per, Lim 1, Fkh, Nvy)

Proposed mechanism for midline VGlut transcriptional regulation GABAergic i. VUM/p. MNB VGlut RNA pol II Sim En H-sib CRM m. VUM CRM 1) En mutants exhibit ectopic VGlut expression in Sim+ neurons 2) En misexpression in H-cell sib represses VGlut

Proposed mechanism for midline VGlut transcriptional regulation GABAergic i. VUM/p. MNB VGlut RNA pol II Sim En H-sib CRM m. VUM 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 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 En En Sim

Transgenic expression analysis Sim En En En Sim 2 independent insertions No embryonic expression

Transgenic expression analysis 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 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 En En Sim 2 independent insertions Expression not determined

Transgenic expression analysis Sim En En En Sim TA cloned Not subcloned into stinger

Conclusions • ry 75/sim. H 9 allelic combination eliminates late sim expression in midline glia and i. VUMs, Hcell sib, and p. MNB • simlate mutants express wrapper, but may have a MG defect. • simlate mutants exhibit ectopic midline axonal projections to the segmental nerve root (likely i. VUM axons) Future Directions • Manipulate Sim function using misexpression of dominant-negative sim constructs; Per-Gal 4 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 Gad 1 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. •