1. 1.Notch signaling and midline cell development 2.Neuron/glia interactions; nrx

2. Notch signaling is required for glia and the MNB Wild- type Dl 3 / Dl 3 MP3 cells (ple) VUM cells (Tbh) MP1 cells (odd) Glia (wrapper) MNB (wor)

3. Only MP4 progeny are produced in Dl mutant embryos How does this phenotype arise?

4. 1. Mesectoderm specification 2. Cell fate specification Model for early midline development

5. 1. Mesectoderm specification 2. Cell fate specification Models for early Delta phenotype Single division 3 rounds of division and cell death

6. Models for early Delta phenotype Single division 3 rounds of division and cell death Predictions # of MPs present at early stages Pattern of division ~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

7. Experiments: Compare wild type and Dl mutants at the earliest possible stages 1.Fixed samples 2.Live imaging

8. Genetics 1. (f) w; sim-gal4,UAS-tauGFPX(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 copy¼ are Dl[3] ¼ have 2 copies

9. Odd-skipped identifies multiple MP1s in Dl mutants

10. 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

11. MP1 divisions likely occur in close succession Pros OddProsOdd Go to LSM for stack

12. 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?

13. 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

14. 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.)

15. 1.Notch signaling and midline cell development 2.Neuron/glia interactions; nrx