Papers: Aims, techniques and finding - Lecture 6

Question 1

What was the title and aims of the Lamb and Harland study?

Answer 1

Lecture 6

“FGF is a direct neural inducer, which, combined with noggin generates anterior-posterior neural pattern”

• Involvement of FGFs, BMP antagonists to generate the whole anteror posterior pattern of the CNS

Question 2

[Brief] Progression of Experiments of Lamb and Harland

Answer 2

1. Does FGF induce neural tissue without mesoderm and does it determine neural character?
2. How do BMP antagonists interact with FGF signalling?
3. Does FGF act as a morphogen?
4. What is the regional character of the tissue in the middle of the polarised structures?

Question 3

What was the series of experiments used in the Lamb and Hardland study?

Answer 3

1. Does FGF induce neural tissue without mesoderm and does it determine neural character?
   
   1. Animal cap (late blastula -> gastrula (low mg/ca)
   2. FGF
   3. Double in situ: (NRP1//M.actin [neural tissue without mesoderm]; Otx2/Eng2 and HoxB9 [does it detemine neural character]
2. How do BMP antagonists interact with FGF signalling?
   
   1. Instead exposure to FGF/Noggin/FGF+Noggin
   2. Stain for NRP1/M.actin
3. Does FGF act as a morphogen?
   
   1. Inhibit BMP with constant Noggin, titrate FGF conc (2,10,50 ng/ml)
   2. Stain for Otc2, HoxB9
4. What is the regional character of the tissue in the middle of the polarised structures?
   
   1. Add FGF/Noggin/FGF+Noggin
   2. Stain Krox20 and M.actin

Question 4

What was the conclusion of the Lamb and Harland study?

Answer 4

Lecture 6

Able to generate the whole anterior posterior pattern of the CNS with the synergistic interaction of FGF and BMP antagonism

Question 5

What was the title and aims of the Pera paper?

Answer 5

Lecture 6

“Integration of IGF, FGF and anti-BMP signals via Smad1-phosphorylation in nerual induction”

Aims:

• Had been previously demonstrated that IGF induces neural tissue
• How does this fit into the interaction of FGF and anti-BMP signals to pattern the neural tissue

Question 6

What was the progression of experiments of the Pera paper; looking at the integration of IGF, FGF and chordin (first part)?

Answer 6

1. IGF, FGF and chordin increase the size of the neural plate
   
   1. In situ hybridisation with Sox2(eraly neurula stage in the neural plate)/N-tubulin(differentiating neurons in the neural plate)
   2. Injection of Chd, IGF2 or FGF
   3. Expansion of expression
2. Neural induction by Chd requires both IGF and FGF signalling
   
   1. Animal cap explants staining for NCAM (general neural), Rx2a (Anterior neural), a-actin (mesodermal).
   2. Animal caps injected with: Chd (NCAM, Rx2a); Chd +IGFR-MO (lose NCAM, Rx2a); Chd+DN-FGFR4 (lose NCAM, Rx2a); Chd+BMP7 (lose NCAM, Rx2a)

Question 7

What was the progression of experiments in the Pera paper; looking into the hypothesis that Smad1 is an integration node of BMP/FGF/IGF?

Answer 7

1. In situ hybridisation of series of Smad1 mutants
   
   • WT-SMAD
   • LM-SMAD (linker region: S –> A)
   • DM-SMAD (linker region and C-terminal region: S –> A)
   • CM-SMAD (C-terminal region: S –> A)
   1. Validated Smad1 mutants all translated equally
      
      • Epitope tagging
        
        1. Added FLAG tag using recombinant DNA tech
        2. Ran on western blot with an Ab to quantifiably assess translation levels
   2. Stain with N-tubulin (Dorsal marker, differentiating neurons in the neural tissue, expressed with BMP inhibition) and Sizzled (Marker of ventral character, expressed with BMP activity)
      
      • Lm-smad lose D axis, upregulation of sizzled at the expense of N-tubulin expression
2. LM-smad inhibits neural induction in vivo
   
   1. Explant assay: Chd, FGF, IGF in explants with WT- or LM-Smad1
   2. Staining for NCAM, Otx2,Keratin, Ef1a
   3. LM-smad able to block the ability of FGF8,IGF2 to induce neural tissue (NCAM, Otx2)
3. Is the Smad1 linker region phosphorylated in vivo?
   
   1. Western blot WE with WT-, LM-SMAD at 8 (blastula), 10.5, 12.5 (gastrula - FGF signalling)
   2. Measuring Smad1/pSmad1 (motility change), ERK2, pERK2 (antibody)
   3. When FGF active, get upregulation of pERK coinciding with the appearance of a higher molecular weight band in WT not in LM-smad

Question 8

[Brief] Progression of experiments of Pera

Answer 8

1. IGF, FGF and chordin increase the size of the neural plate
2. Neural induction by Chd requires both IGF and FGF signalling
3. In situ hybridisation of series of Smad1 mutants
4. Validated Smad1 mutants
5. LM-smad inhibits neural induction in vivo
6. Is the Smad1 linker region phosphorylated in vivo?