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

Notch signaling and midline cell development Neuron/glia interactions; nrx. Notch signaling is required for glia and the MNB. MP1 cells ( odd ). MP3 cells ( ple ). VUM cells ( Tbh ). MNB ( wor ). Glia ( wrapper ). Wild- type. Dl 3 / Dl 3.

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

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

  2. Notch signaling is required for glia and the MNB MP1 cells (odd) MP3 cells (ple) VUM cells (Tbh) MNB (wor) Glia (wrapper) Wild- type Dl3 / Dl3

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

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

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

  6. Models for early Delta phenotype 3 rounds of division and cell death Single division Predictions # of MPs present at early stages ~15; 5 each of MP1, MP3, and MP4 ~3; 1 each of MP1, MP3, and MP4 Pattern of division Multiple divisions from s11 to s14 All divisions in en masse at s11

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

  8. 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

  9. Odd-skipped identifies multiple MP1s in Dl mutants

  10. Odd and Cas identify 3 distinct MP populations in Dl mutants OddCas • Tentative conclusions: • ~5 each of MP1, MP3, and MP4 are specified in Dl mutant embryos. • Additional experiments: • Look at more segments stained with Odd and Cas • Ask if the MP6 is present by staining with Cas and Tkr (likely Cas- Tkr+). • Establish pattern of MP division using pH3, Odd, Cas and sim-Gal4 UAS-tauGFP as well as using live imaging. Go to LSM for stack

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

  12. Tentative model 1. Mesectoderm specification • Some additional questions: • Which cell becomes the MP? What are the txn factors and/or signaling that selects the MP? • Candidate genes – odd, cas • How are the MP5, MP6, and MNB specified? • How are non-MP cells directed to the AMG or PMG fate? 2. Cell fate specification equivalence groups

  13. What to do next • hedgehog and wingless are doing something, what is it? • Maybe required to confer anterior and posterior identity (ie. MP1,3,4 vs MP5,6, MNB or AMG vs PMG) • phenotypic analysis of wg and hh mutants • misexpression of constitutively active downstream effectors (Tcf.VP16 , Ci.VP16, and others). • Reporters of activity (nuclear arm, ptc expression)

  14. What to do next (cont.) • How is glial gene transcription regulated? • What are the dynamics of glial transcription? • examine the expression patterns of genes with de novo expression in midline glia (~17 genes) • Identify temporal and spatial patterns that may give clues to regulatory hierarchy. • What is the role of Notch signaling? • isolate enhancers for several glial genes and mutate binding sites for Suppressor of Hairless. • Examine expression in Dl mutants. • What is the role of single-minded? • isolate enhancers for several glial genes and mutate binding sites for Sim. • Examine expression of reporters in sim mutants.

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

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