1. Notch signaling and midline cell development2. Neuron/glia interactions; nrx

Notch signaling is required for glia and the MNBW

ild- t

ype

Dl3 /

Dl3

MP3 cells (ple)

VUM cells(Tbh)

MP1 cells (odd)

Glia(wrapper)

MNB(wor)

Only MP4 progeny are produced in Dl mutant embryos

How does this phenotype arise?

1. Mesectoderm specification

2. Cell fate specification

Model for early midline development

1. Mesectoderm specification

2. Cell fate specification

Models for early Delta phenotype

Single division 3 rounds of division and cell death

Models for early Delta phenotype

Single division 3 rounds of division and cell death

Predictions

# of MPs presentat early stages

Pattern ofdivision

~15; 5 each of MP1, MP3, and MP4 ~3; 1 each of MP1, MP3, and MP4

All divisions in en masse at s11 Multiple divisions from s11 to s14

Experiments:Compare wild type and Dl mutants at the earliest possible stages

1. Fixed samples

2. Live imaging

Genetics

1. (f) w; sim-gal4,UAS-tauGFP X (m) Dl[3]/TM6b

sim-gal4, UAS-tauGFP/+ ; Dl[3]/+ or TM6b/+

2. (f,m) sim-gal4, UAS-tauGFP/+ ; Dl[3]/+

sim-gal4, UAS-tauGFP/ sim-gal4, UAS-tauGFP ; Dl[3]/Dl[3](sim-gal4, UAS-tauGFP/+)x2 Dl[3]/+ +/+ +/+

¾ progeny have at least 1 copy ¼ are Dl[3]¼ have 2 copies

Odd-skipped identifies multiple MP1s in Dl mutants

Odd Cas

Go to LSM for stack

Tentative conclusions:

1. ~5 each of MP1, MP3, and MP4 are specified in Dl mutant embryos.

Additional experiments:

1. Look at more segments stained with Odd and Cas

2. Ask if the MP6 is present by staining with Cas and Tkr (likely Cas- Tkr+).

3. Establish pattern of MP division using pH3, Odd, Cas and sim-Gal4 UAS-tauGFP as well as using live imaging.

Odd and Cas identify 3 distinct MP populations in Dl mutants

MP1 divisions likely occur in close succession

Pros Odd Pros Odd

Go to LSM for stack

Tentative model

1. Mesectoderm specification

2. Cell fate specification

equivalence groups

Some additional questions:1. Which cell becomes the MP? What

are the txn factors and/or signaling that selects the MP?Candidate genes – odd, cas

2. How are the MP5, MP6, and MNB specified?

3. How are non-MP cells directed to the AMG or PMG fate?

1. hedgehog and wingless are doing something, what is it?a. Maybe required to confer anterior and posterior identity (ie.

MP1,3,4 vs MP5,6, MNB or AMG vs PMG)

b. phenotypic analysis of wg and hh mutants

c. misexpression of constitutively active downstream effectors (Tcf.VP16 , Ci.VP16, and others).

d. Reporters of activity (nuclear arm, ptc expression)

What to do next

2. How is glial gene transcription regulated?a. What are the dynamics of glial transcription?

i. examine the expression patterns of genes with de novo expression in midline glia (~17 genes)

ii. Identify temporal and spatial patterns that may give clues to regulatory hierarchy.

b. What is the role of Notch signaling?i. isolate enhancers for several glial genes and mutate binding

sites for Suppressor of Hairless.

ii. Examine expression in Dl mutants.

c. What is the role of single-minded?I. isolate enhancers for several glial genes and mutate binding

sites for Sim.

II. Examine expression of reporters in sim mutants.

What to do next (cont.)

1. Notch signaling and midline cell development2. Neuron/glia interactions; nrx