Papers: Aims and experiments - lecture 4

Question 1

What were the papers involved in looking at Vg1?

Answer 1

1. Rebagliati - “Idenitification and cloning of localised maternal RNAs from Xenopus eggs”
2. Weeks and Melton (Thomas and Moos) - “A Maternal mRNA Localized to the Vegetal Hemisphere in Xenopus Eggs Codes for a Growth Factor Related to TGFbeta”
3. Dale - “Secretion and mesoderm-inducing activity of the TGF-b-related domain of Xenopus Vg1”
4. Birsoy - “Vg1 is an essential signaling molecule in Xenopus development”

Question 2

What was the title and aim of the Rebagliati study?

Answer 2

“Idenitification and cloning of localised maternal RNAs from Xenopus eggs”

Identifying a maternally depositied RNA that is localised to the vegetal hemisphere

Question 3

[basic] Progression of experiments of the Rebagliati study?

Answer 3

1. What factors are different in the animal/vegetal hemisphere before mid-blastula transition (differential screen)
2. Independent assays of the localisation of VG1 (spacial/time)

Question 4

What was the title and aim of the weeks and melton paper?

Answer 4

“A Maternal mRNA Localized to the Vegetal Hemisphere in Xenopus Eggs Codes for a Growth Factor Related to TGFbeta”

1. Vg1 member of the TGFb family
2. Formation of mesoderm is specified by VG1

Question 5

[basic] Progression of the experiments of Weeks and Melton?

Answer 5

1. Characterisation of VG1

2. VG1 in Xenopus oocyte tightly localised to vegetal cortex

Question 6

What did Thomas and Moos show?

Answer 6

VG1 in the Xenopus blastula

Distributed in the vegetal hemisphere

Question 7

What was the title and aim of the Dale paper?

Answer 7

“Secretion and mesoderm-inducing activity of the TGF-b-related domain of Xenopus Vg1”
Synthetic VG1 has no effect on development, does not induce mesoderm in animal caps, doesn’t form dimers, isn’t secreted.
BMP4-VG1 fusion protein does.

Role for VG1 in mesoderm induction in xenopus development.

Question 8

[basic] progression of experiments of the Dale paper

Answer 8

1. VG1 neither processed nor secreted in oocytes
2. VG1 does not form dimers
3. Synthetic VG1 has little biological activity
4. BMP-VG1 is processed and forms dimers in oocytes
5. BMP-VG1 secreted by oocytes
6. BMP-VG1 mRNA induces mesoderm

Question 9

What was the title and aim of the Birsoy paper?

Answer 9

“Vg1 is an essential signalling molecule in Xenopus
development”
VG1 essential for xenopus embryonic development, mesoderm induction and the expression of some key BMP-antagonists
Second allele of VG1 effective at rescuing VG1 deleted embryos

Question 10

[basic] Progression of experiments in the Birsoy paper?

Answer 10

1. Knocked down VG1 with inhibitory reagents
2. Validation of PO (real-time PCR) and MO (western blot)
3. Biological effect of knocking down (reduced heads/gastrulation and effect on organiser markers cerb/chd) with PO
4. Western blot of pSmad2/1 with PO
5. VG1 in mesoderm induction in vivo
6. Western blot with VG1(S/P) to show processing difference
7. VG1(S) can rescue effect of VG1 depletion