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Fig. 3. Tril is required upstream and
downstream of Smad1 phosphorylation for
Bmp target gene expression. (A) β-
galactosidase (lacZ) RNA was co-injected with
control or Tril MOs (40 ng) as illustrated. Embryos
were stained for β-galactosidase activity at stage
11 to identify the half of the embryo that received
the MOs (indicated by a red bracket) and
expression of Bmp target genes was analyzed by
WMISH. (B-E) Tril, control (Con) or no (None)
MOs were injected into two-cell embryos.
(B) Expression of msx1, ventx2, szl and odc
analyzed by RT-PCR. Results were reproduced in
three experiments. -RT, no reverse transcriptase in
cDNA synthesis reaction. (C,D) Levels of pSmad1/
5/8, total Smad1/5/8 (tSmad1/5/8) and β-Actin
were analyzed by immunoblot at the indicated
stages. A representative blot (D) and normalized
data (levels of Smad1/5/8 normalized to β-Actin)
from three independent experiments (values are
mean±s.d.) (C) are shown. (E) The indicated
tissues were explanted from 10 embryos in each
group and levels of pSmad1/5/8, tSmad1/5/8 and
β-Actin analyzed by immunoblot. Results were
reproduced in two experiments. (F) Tril or control
MOs were injected alone, or together with RNA
encoding caSmad5, into both dorsal cells of fourcell
embryos. The dorsal quadrant of the embryo
was explanted at stage 10 and cultured until stage
11, at which time expression of t, msx1 and odc
was analyzed by RT-PCR. Results were
reproduced in three independent experiments.
(G) Tril functions both upstream and downstream
of pSmad1/5/8 to enhance Bmp signaling. |
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Fig. 2. Tril is required in ectodermal and
mesodermal cells for blood commitment.
(A,B) Control or Tril MOs (40 ng) were injected into both
ventral cells of 4-cell embryos and expression of
hba3.L, scl or gata1 was analyzed by WMISH at stage
34 in three experiments. hba3.L staining in the
posterior VBI (pVBI) was scored as absent (0+), weak
(1+), very weak (2+) or strong (3+), as illustrated. aVBI,
anterior VBI. (C) Control or Tril MO1 (40 ng) and/or tril
RNA lacking the 5′UTR (100 pg) was injected into two
ventral vegetal blastomeres of eight-cell embryos and
expression of hba3.L was analyzed by northern blotting
at stage 34. Levels of hba3.L transcripts normalized to
odc and reported as a percentage of hba3.L levels in
control embryos are shown below each lane. Results
were replicated in three experiments. (D) Control or Tril
MO1 (40 ng) was injected into both cells of two-cell
embryos and expression of scl was analyzed by RTPCR
at stage 15. Results were reproduced in three
experiments. -RT, no reverse transcriptase in cDNA
synthesis reaction. (E) Control or Tril MO1 (40 ng) was
injected into two-cell embryos. At stage 10, ectoderm
(ecto) or ventral mesoderm (VM) was explanted and
recombined in the combinations indicated below the
graph on the right. Expression of hba3.L was analyzed
in 10 pooled recombinants in each group (left) or in 10
whole sibling embryos (right) at stage 34 by qPCR
(mean±s.d., data analyzed by two-tailed t-test).
Results were reproduced in two independent
experiments. |
