Download emboj7601684-sup

Supplementary Figure Legends
Supplementary Figure 1
Structural comparison of Tsh-Family proteins.
(A) The domain structures of XTsh3, mouse Tsh1-3 (mTsh1-3), fly Tsh and fly Tiptop are
shown. Blue, Zn-finger motif; gray, Hox domain; black, basic motif. The percentage of
identical amino acid residues (compared to the corresponding domain of XTsh3) is shown
above each domain. The amino-acid residues in the basic motif of XTsh3 that are
conserved among the vertebrate proteins are underlined. The basic residue-rich motifs in
fly proteins (indicated by asterisks) are not conserved with the vertebrate basic motifs in
primary sequences. (B) The amino- and carboxyl-terminal mutants of XTsh3 (XTsh3-N
and C). The break point for the deletion is at amino acid reside 399.
Supplementary Figure 2
MO-mediated loss-of-function analysis.
(A) Temporal profile of XTsh3 expression during development analyzed by quantitative
RT-PCR. (B) Design and sequences of Tsh3-MO-A and its control MO. (C) Design and
sequences of XTsh3-MO-C and its control MO. (D) Western blot analysis of
epitope-tagged XTsh3 proteins produced in the RNA-injected stage 10.5 embryos.
Coinjection of XTsh3-MO-A or that of XTsh3-MO-C inhibited the production of
epitope-tagged XTsh3 proteins. Control MOs (5-base-mispaired XTsh3-MO-A and
XTsh3-MO-C) did not affect the translation. HSP70, loading control. (E,F) Quantitaitve
RT-PCR analysis of DV markers in XTsh3MO (E)- or control MO (F)-injected embryos
(stage 11). (G,H) Regulation of Xnr-3 (Wnt target gene) expression by XTsh3. (G)
Reduction of Xnr-3 expression by XTsh3MO injection in stage 11 embryos. (H) Induction
of Xnr3 in XTsh3-injected animal caps (stage 11 equivalent). (I-L) Rescue experiments of
Myf5 and Gsc expression. XTsh3-MO-C (10 ng/cell) was used. (I) Reduced Myf5
expression in the XTsh3MO-injected embryo (76%, n = 17). (J) Recovery of Myf5
expression by co-injection of XTsh3 (the coding region only; reduction in 8%, n = 12). (K)
Reduced Gsc expression in the XTsh3MO-injected embryo (reduction in 41%, n = 17) (L)
Recovery of Gsc expression by co-injection of XTsh3 (reduction in 16%, n = 12). (M-P)
Use of the LacZ tracer for confirming dorsal and ventral injections. (M,N) Injection of
XTsh3-MO and LacZ RNA into two dorsal (M) or ventral (N) blastomeres at the 4-cell
stage. (O,P) Injection of Dsh and LacZ into a ventral blastomeres at the 8-cell stage with
-1-
(O) or without (P) XTsh3.
Supplementary Figure 3
Ventralized phenotype in stage 10.5 embryos injected with XTsh3-MO-C (B,D,F,H; radial
injection at 10 ng/cell, 4-cell stage). MO-injection caused suppression of the dorsal genes
Chd, gsc and upregulation of the ventral genes Szl ad Xvent1, showing that two
XTsh3-MOs have similar effects on DV patterning.
Supplementary Figure 4
XTsh3 specifically interacts with Wnt/ß-catenin signaling.
(A,B) XTsh3 overexpression did not increase FOPflash (control mutant for
TOPflash)-luciferase activity either in animal caps (A) or HEK293T cells (B). (C,D)
Quantitaitve PCR analysis of the expression of the Activin target genes Xbra (C) and
Mix2 (D) in the animal cap (stage 10 equivalent). Unlike Activn treatment (2 ng/ml on
stage 8.5 animal cap for 3 hours), XTsh3 substantially induce neither genes. (E) XTsh3-C
suppressed TOPflash activity elevated by Wnt signaling in the animal cap. The
experimental conditions are the same as in Figure 4. (F) The presence of low (but
significant) basal ß-catenin-dependent activity of TOPflash reporter in the animal cap
(stage 11). The basal TOPflash reporter activity (cont, left column) was lowered by
injection of ß-catenin-MO (right column). Such reduction was not seen with the FOPflash
activity (not shown).
Supplementary Figure 5
Nuclear localization of XTsh3 requires its amino-terminal portion.
Immunostaining analysis of the subcellular localization of epitope-tagged XTsh3 and its
deletion mutant proteins in the RNA-injected animal cap (stage 11). XTsh3 and
XTsh3-C were located specifically in the nucleus (A,C), whereas XTsh3-N was
detected in the cytoplasm and the submembrane region (E). DAPI, nuclear staining
control.
Supplementary Figure 6
Nuclear localization of XTsh3 is not affected by augmented or attenuated Wnt signaling.
-2-
Immunostaining analysis of the subcellular localization of epitope-tagged XTsh3 with
increased (I) or decreased (E,G) Wnt signaling. In all cases, XTsh3 was localized
specifically to the nucleus. DAPI, nuclear staining control.
Supplementary Figure 7
Control of dorsalization by XTsh3 and Wnt signaling.
(A) RT-PCR analysis of VMZ explants. The ventral-most part of the MZ (60 degree) was
excised from the stage 10.5 embryo, cultured in 1x LCMR, and analyzed at the equivalent
of stage 13. The dorsal marker Chd was induced at a low level and the lateral and ventral
markers Wnt8 and Vent2 were moderately attenuated. It appears that XTsh3 could induce
only weak dorsalization in the VMZ. (B-D) XTsh3-MO decreased the secondary axis
formation induced by a moderate dose of ß-catenin. (B) Control. (C) Double axis
phenotype induced by a single ventral-vegetal injection of ß-catenin RNA (20 pg, 8-cell
stage; double axis in 44%, n=18). (D) Coinjection of XTsh3-MO with ß-catenin (double
axis in 13%, n=54). (E,F) Radial expansion of the dorsal marker Chd (E) by radial
injection of a high dose of ß-catenin (100 pg/cell x 4 cells). Coinjection of XTsh3-MO (20
ng/cell; F) did not suppress Chd expression caused by this high dose of ß-catenin. (G-L)
Effect of XTsh3-MO on nuclear ß-catenin in the dorsal marginal zone region at stage 10.
Embryos were injected with control (G-I) or XTsh3-MO (J-L). (G,J) Fluorescent
immunostaining of endogenous ß-catenin. (H,K) Nuclear DAPI staining. (J,L) Merged
pictures of the ß-catenin and DAPI signals. Open arrowhead, the dorsal lip. (M)
Quantitative RT-PCR analysis of maternal XTsh3 in the dorsal and ventral blastomeres of
16-cell embryos. Wnt 11 was used as control.
Supplementary Figure 8
XTsh3-C causes phenotypes opposite to those induced by the wild-type XTsh3.
(A) Whole-mount in situ hybridization of the dorsal genes Chd (top row) and gsc (middle
row) and the ventral gene Xvent2 (bottom row) in control (left column), XTsh3-N
(middle column) or XTsh3-C-injected (right column) embryos. XTsh3-C injection
caused suppression of the dorsal gene expression and upregulation of the ventral gene.
(B) Immunostaining study of nuclear ß-catenin localization in animal cap cells. Animal
caps (excised at stage 9 and fixed at stage 11) were prepared from embryos injected with
control (top row), Wnt1 (middle row), Wnt1 and XTsh3-C (bottom row). (left column)
-3-
Fluorescent immunostaining of endogenous ß-catenin. (middle column) Nuclear DAPI
staining. (right column) Merged pictures of the ß-catenin and DAPI signals. Strong
nuclear ß-catenin signals were found in 8%, n=138 nuclei for control, 64.4%, n=149 for
Wnt-injected, 12.7%, n=142 for Wnt + XTsh3-C-injected animal caps.
-4-