Building a functional nervous system Immature CNS Cells Motorneurons Neurosecretory cells Glia Interneurons Cell fate determination Cell migration Apoptosis Axonogenesis Synapse formation Ensheathment

Big questions 1.How do MG arise? a.transcriptional control b.formation/ fate (hh signaling) 2.What are MG doing?

2 types of MG are present at stage 10

Notch signaling is required for MG cell fate wrapper Dl 3 / Dl 3 Sim-Gal4> UAS-Su(H).VP16 wild type MP MG Delta  Notch  Su(H)  MG gene expression

What is the glial gene expression hierarchy? Are there multiple mechanisms for MG gene expression? How are alternate mechanisms used? shadow enhancers? as parts of an overall expression pattern? How are PMG and AMG specified differentially? MG gene expression Notch Su(H) Delta ????? DVvl Sim

When does MG gene expression begin? s9s10s11s12>s12 CG32244-P A/P A/P A/P wrapper-AA/ P A/ P A/ P epac--A/PA/PA/P CG AANA argos--PA/PA/P CG P P NA CG AA CG AA CG AA CG AA netANA netBNA pqbp-1NA shepNA sim(2.8kb frag)--?A/PA/P slit-lacZ--?A/PA/P tslNA wNA

MG gene expression argos CG31145 epac AMG only PMG only AMG and PMG

summary 1.The timing of the initiation of MG gene expression suggests that there may be multiple mechanisms for turning on MG gene expression.

Future direction 1.Examine the expression of additional MG expressed genes during stages Test enhancer fragments for MG gene expression 3.Identify potential transcription factor binding sites in MG enhancer fragments a.Test the requirement for binding sites by site directed mutagenesis b.Examine enhancer fragment expression in mutants How does the regulation of the de novo set of genes compare with the non de novo set identified by Joe Pearson

Big questions 1.How do MG arise? a.transcriptional control b.formation/ fate (hh signaling) 2.What are MG doing?

Hh signaling, in brief Ptc Hh Smo CiActCiRep Ptc Smo Hh CiActCiRep Other Proteins Nucleus Cytoplasm

hh directs midline neuronal fate wild type hh- AA142-lacZ (MG) Hummell et al., 1999 X55-lacZ (MN) ptc- 16 cells/seg2-4 cells/seg 0-1 cell/seg ># of cell/seg constitutive repression of hh targets. constitutive activation of hh targets

Where is hh expressed?

hh specifies posterior cell fates Bossing and Brand., 2006 in hh mutant: at stage 10: en and l(1)sc expression are lost in the midline (~2 en+ cells/seg) at stage 13: en expression is lost (i.e. VUM neurons are missing) sim-gal4>UAS-en results in a loss of MP1 neurons Therefore: hh  en in the posterior which induces posterior cell fate

How does this correspond to what we know?

Model 1: hh specifies posterior cell fates anterior fatesposterior fates hh Outcome of hh mutant: In the absence of hh function, posterior fates (MP4-6, MNB, and PMG) are transformed into anterior fates (MP1, MP3, AMG). Would see increase in MP1 and MP3 neurons and ~ 10 MG per segment This is inconsistent with Hummell data where there is a reduction of X55+ neurons and increase in AA142+ MG.

Model 2: hh regulates all MP formation PMG Outcome of hh mutant: In the absence of hh function, all MPs are transformed into MG). ~16 MG/ segment This is consistent with Hummell data where there an increase to 16 AA142 cells per segment. Reports of loss of sim expression in hh-. Maybe because there is not Notch signaling from MPs. AMG PMG AMG

Model 3: hh regulates posterior cell formation PMG Outcome of hh mutant: In the absence of hh function, MP4-6, MNB would not develop properly (maybe transformed into PMG). ~13 MG/ segment MP1,3,4 would be present. This is sort of consistent with Hummell data. > MG < MN

Experiments 1.loss of function a.hh mutants (AC: p-element deletion, 13C: strong ems allele) b.cross into sim-Gal4 UAS-tauGFP background (use to count midline cells) c.examine markers for midline cell fate at stage (use to identify cell types) 2.misexpression using sim-Gal4, UAS-tauGFP a.UAS-hh – activate hh targets in all midline cells b.UAS-hhN – activate hh targets in all midline cells c.UAS-Ci[76] – represses hh targets in all midline cells 3.Reporter expression a. ptc-lacZ – activated in response to hh signaling

A tiny bit of preliminary data Sim stage 15 sagittal hh[AC]/ hh[AC]Sim stage 15 sagittal hh[AC]/ +