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 controlb. 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

wra

pper

Dl3 / Dl3Sim-Gal4>

UAS-Su(H).VP16wild type

MP

MG

MG MG

MGMG

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

?????D VvlSim

When does MG gene expression begin?

s9 s10 s11 s12 >s12CG32244 - P A/P A/P A/Pwrapper - A A/P A/P A/P

epac - - A/P A/P A/PCG31145 - - A A NAargos - - P A/P A/PCG31116 - - P P NACG32030 - - - A ACG33275 - - - A ACG7271 - - - A ACG8776 - - - A AnetA NAnetB NApqbp-1 NAshep NAsim (2.8kb frag) - - ? A/P A/Pslit-lacZ - - ? A/P A/Ptsl NAw NA

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 10-12

2. Test enhancer fragments for MG gene expression

3. Identify potential transcription factor binding sites in MG enhancer fragmentsa. Test the requirement for binding sites by site directed

mutagenesisb. 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 controlb. formation/ fate (hh signaling)

2. What are MG doing?

Hh signaling, in brief

Ptc

Hh

Smo

CiAct CiRep

PtcSmo

Hh

CiAct CiRep

Other Proteins 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/seg 2-4 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., 2006in 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 fates posterior 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 neuronsand ~ 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

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 PMGPMGAMG

Model 3: hh regulates posterior cell formation

PMG

PMG

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 functiona. 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 10-17 (use to identify cell types)

2. misexpression using sim-Gal4, UAS-tauGFPa. UAS-hh – activate hh targets in all midline cellsb. UAS-hhN – activate hh targets in all midline cellsc. UAS-Ci[76] – represses hh targets in all midline cells

3. Reporter expressiona. 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]/ +