| |
Fig. 6. ASPN inhibits multiple signal molecules. (A) ASPN
blocks endogenous Activin, BMP and Wnt signals, as examined
by luciferase assays. ARE-luc, BRE-luc or TOPFLASH reporter
constructs were injected with 1 ng β-Galactosidase (control),
100 pg Xnr1 mRNA (for ARE), 100 pg BMP4 mRNA (for
BRE),100 pg Wnt8 mRNA (for TOPFLASH), 100 pg Xnr1+1 ng
ASPN mRNAs (for ARE), 100 pg BMP4+1 ng ASPN mRNAs (for
BRE) or 100 pg Wnt8+1 ng ASPN mRNAs (for TOPFLASH) and
were assayed at stage 12. (B) ASPN inhibits the Nodal signalling
pathway. Animal caps injected with control or ASPN mRNA were
prepared at stage 9 and cultured with control medium or medium
containing human Nodal protein until stage 10.5. Mix.2
expression was analysed by qRT-PCR. (C,D) Xbra expression
was inhibited by ASPN, as analysed by in situ hybridisation. The
β-Galactosidase mRNA (light blue product) was injected without
(C) or with (D) ASPN mRNAs into one blastomere at the equator
region of 4-cell stage embryos and embryos were cultured until
stage 10.5. Affected areas are indicated with arrowheads.
(E) ASPN has neural-inducing activity. Animal caps injected with
500 pg Chd (lane 3) or 1 ng ASPN (lane 4) mRNAs were
analysed at stage 14 by semi-quantitative PCR. (F) ASPN inhibits
the Wnt signalling pathway. Animal caps injected with Wnt8 and
ASPN mRNAs were prepared and the expression of Xnr3 was
analysed at stage 10.5. (G-I) ASPN forms complexes with BMP4
(G), Xnr1 (H) and Wnt8 (I) proteins. In order to avoid artificial
interactions in the same cells, each expression construct was
separately transfected into HEK293 cells and cells were
combined on the following day as indicated. The cell lysates were
collected after two additional days and immunoprecipitation (IP)
was performed with the HA antibody and western blotting (IB) was
performed with the FLAG (G) or myc (H,I) antibodies (*P<0.01;
**P<0.05; Student’s t-test). Error bars represent s.e.m. |
