Cerebus paper exam 1.txt

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  1. Cerebus role
    • Part of TGF-B family
    • Expressed during gastrulation: Encodes a protein that is responsible for induction of ectopic heads in absence of trunk. Acts as an antagonist to Wnt, BMP and Nodal proteins by binding to them at INDEPENDENT sites
    • Inhibition of trunk mesoderm
    • Neutralization of ectoderm
    • Expressed dorsally
  2. Cerebus Fig 1
    • Main point: Cer-L is able to bind to Xnr-1, BMP and Xwnt-8 at individual sites in solution
    • First Cer tagged with FLAG: 2 products found, Cer-S and Cer-L
    • Looked at binding of Cer to mesoderm inducing factors Xnr1, Vg1 and Activin: IP showed Xnr1 binds, not the other 2
    • Then looked at Cer in presence of all 3 above, and saw that Xbra was decreased with only Xnr1 present: Cer inactivates Xnr1 signaling
    • Cer-L (not Cer-S) tagged with FLAG showed binding to BMP4 (Blocks Cer binding) AND also NEUTRALIZATION activity, whereas Cer-S did NOT
    • FULL length cer –mRNA INHIBITS Xwnt8 protein, and to test if the binding of Xwnt to Cer is extracellular, Flag-tagged CerS and Cer-L and then added Xwnt-8-HA protein and saw IP with Cer-L
    • Xnr1 binding NOT displaced by BMP4, and Wnt NOT displaced by excess BMP4 Xnr1, so binds at independent sites to all 3 signaling factors
  3. Cerebus Fig 2
    • Main point: Cer-S inhibits nodal related signals to lead to ectopic head formation
    • Xenopus embryo used to see if Cer-S mRNA injected causes blocking of expression: Mesoderm markers Xnr1 and Xwnt-8 BLOCKED with Cer.
    • Expression of endogenous cer was BLOCKED by cer-smRNA
    • Nodal signal Xnr1 induced endogenous cerebus, but only early on (RNA level), not at the DNA level of Xnr1
    • Looked at BMP signals: tBR blocks BMP signals, and when this occurred with cer-S, head like formation occurred *similar to when full length cer inserted.
    • Did not do anti-Wnt activity experiments, except for looking at loss of Xwnt in embryos in beginning figs.
  4. Nodal activity sufficient in Cer-S
    NOT Sufficient to induce head-like structures alone
  5. Fig 3:
    • Main point: All three inhibitory activities of cerebus are REQUIRED for ectopic head formation
    • Looked at whole embryos and added antagonists
    • Chd: Anti-BMP
    • Cer-s: anti-nodal
    • Adding chd and cer-s: able to see ectopic head structures
    • When they added Xwnt-8 DNA ectopic head BLOCKED
    • ALK-4 receptor: blocks anti-nodal activity of cerebus mRNA, resulted in ABOLISHED ectopic head formation
    • BMPR: BMP receptor, when added it stopped ectopic head formation
    • Frzb-1: anti-Wnt (inhibitor for Wnt protein)
    • When chd (anti-BMP) and frzb-1 were added, ectopic head formation occurred
    • Xnr-1 DNA added (nodal signal)
  6. Role of anterior endoderm with regards to head development (chart)
    • Early endoderm signals Vg1 and Nodal come in, as well as antagonists chordin(a-BMP), Frzb1 (a-wnt) activate Cerebus during gastrulation.
    • Head formation occurs only if all three (Nodal, Wnt and BMP) proteins are inhibited.
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Cerebus paper exam 1.txt
2013-10-09 00:22:34

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