| |
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. |
