June 15, 2007;
Mouse homologues of Shisa antagonistic to Wnt and Fgf signalings.
In an effort to identify Otx2
targets in mouse anterior neuroectoderm
we identified a gene, mShisa, which is homologous to xShisa1 that we previously reported as a head
inducer in Xenopus. mShisa encodes an antagonist against both Wnt and Fgf signalings; it inhibits these signalings cell-autonomously as xShisa1 does. The mShisa expression is lost or greatly reduced in Otx2
mutant visceral endoderm
, anterior mesendoderm
and anterior neuroectoderm
. However, mShisa mutants exhibited no defects in head
is composed of five subfamilies, but normal head
development in mShisa mutants is unlikely to be explained in terms of the compensation of mShisa deficiency by its paralogues or by known Wnt antagonists in anterior
and/or anterior mesendoderm
[+] show captions
mShisa functions antagonistic to Wnt and Fgf signalings. (A, B) Expansion of Otx2-positive anterior neuroectoderm by mShisa overexpression (n = 19/26) in Xenopus embryos. lacZ mRNA (100 pg) was unilaterally injected into two right blastomeres at the four-cell stage (red) either with (B) or without (A) mShisa RNA (100 pg). The embryos were hybridized at stage 14 with Otx2 (blue); whole-mount dorsal views are shown. (C, D) Enlargement of cement gland and anterior structure by mShisa overexpression (n = 25/28). mShisa RNA (100 pg) was injected into animal portion of each blastomere at the 4-cell stage (D). Lateral views of the embryos at stage 23 are shown. (E�G) Inhibition of Wnt and Fgf signalings by mShisa overexpression in Xenopus animal cap. (E) xWnt8 (1 pg) mRNA was injected either alone (lane 3) or together with mShisa mRNA (100 pg for lane 4 and 200 pg for lane 5) into each animal blastomere at the four-cell stage. RNAs were isolated from animal cap explants at late blastula, and Xnr3 expression was analyzed by RT-PCR. Lane 2 the expression with RNAs from animal cap explant not injected with xWnt8. (F) Animal caps were isolated at stage 9 from mShisa mRNA injected (50 pg for lane 4 and 100 pg for lane 5) or uninjected (lanes 2, 3) embryos and cultured without (lane 2) or with (lane 3�5) 50 ng/ml bFgf (R & D Systems) in LCMR for 3 h, and Xbra expression was analyzed by RT-PCR. (G) Animal caps were isolated at stage 9 from mShisa mRNA injected (50 pg for lane 4 and 100 pg for lane 5) or uninjected (lanes 2, 3) embryos and cultured without (lane 2) or with (lanes 3�5) 2.5 ng/ml Activin (R&D Systems) in LCMR for 3 h, and Xbra and Mix2 expression was analyzed by RT-PCR. Lane 1 gives each expression with RNAs from normal stage 11 whole embryos (WE). Histon H4 (H4) expression serves as the RT-PCR control. The − RT panels indicate the PCR for H4 expression with cDNAs synthesized without reverse transcriptase. (H�K) ER localization of mShisa. Hek293T cells transfected with mShisa tagged with HA were stained for HA (green in panel I) and an ER marker calreticulin (red in panel J). (K) gives the merged view of these two signals. (L) Cell-autonomous suppression of Wnt signaling. Ligand cells (S-cells) expressing Wnt3a and receptor cells (R-cells) expressing Fz8 and Lrp6 together with TOPFLASH reporter were prepared separately and cocultured for stimulation of Wnt signaling. When mShisa was co-expressed with Wnt ligand in S-cells, reporter activity was not affected (lanes 5, 6); however, mShisa expression in R-cells suppressed the reporter activity below the basal level (lanes 3, 4). Cells were transfected in 24-well plate with DNAs: Wnt3a, 200 ng; Fz8, 5 ng; Lrp6, 5 ng; mShisa, 50 ng or 200 ng.