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Fig. 5.
PN1 functions in an HtrA1-, FGF- and Wnt-dependent manner. (A-D′) Lateral view of early gastrula embryos. PN1-MO, HtrA1 mRNA and Fgf4 mRNA, but not co-MO, induce ectopic Xbra expression (A-D) and a reduction of Foxi1e expression (A′-D′) in the animal hemisphere. (E-F′) In PN1 morphant embryos, 20 ng HtrA1-MO and XFD mRNA restore normal expression of Xbra and Foxi1e. (G-I) Random-MO, PN1-5mm-MO, and a combination of PN1-MO and non-targeted FLAG-PN1 mRNA do not affect Xbra expression. (J-T) Dorsal view of neurulae. A single injection of PN1-MO shifts the border between Otx2 and Papc (brackets in K) and expands Xcad3 expression (arrowheads in T) anteriorward. Co-MO and PN1-5mm-MO have no effect on Otx2 and Papc (J,R), and random-MO has no effect on Xcad3 expression (S). HtrA1 mRNA, Fgf4 mRNA and Wnt3a-DNA reduce Otx2, but only HtrA1 and Fgf4 expand Papc expression (L,M,P). Co-injections of 5 ng HtrA1-MO, XFD and Dkk1 mRNA revert the effect of PN1-MO and cause slight expansion of Otx2 and posteriorward retraction of Papc signals (N,O,Q). (U-BB) PN1-MO reduces head structures (arrowhead) and expands the proctodeum (bracket in V), whereas co-MO, random-MO and PN1-5mm-MO have no effect in tailbud embryos (U,AA,BB). 20 ng HtrA1-MO, XFD, Dkk1 and FLAG-PN1 rescue posteriorization in PN1 morphants (W-Z). Injected mRNA amounts per embryo were: HtrA1, 200 pg (50 pg in L); Fgf4, 2 pg (0.5 pg in M); XFD, 80 pg (20 pg in O); FLAG-PN1, 800 pg; Dkk1, 24 pg (8 pg in Q). Indicated phenotypes were shown by: A, 136/144; A′, 50/59; B, 154/186; B′, 50/69; C, 21/21; C′, 47/57; D, 79/79; D′, 93/97; E, 66/90; E′, 52/56; F, 72/83; F′, 45/51; G, 24/30; H, 44/46; I, 14/24; J, 12/13; K, 14/14; L, 60/60; M, 45/49; N, 17/17; O, 9/9; P, 34/36; Q, 55/55; R, 31/32; S, 8/8; T, 28/30; U, 75/77; V, 97/95; W, 48/59; X, 23/24; Y, 67/65; Z, 22/23; AA, 7/10; BB, 10/15. |
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Fig. S4. PN1 inhibits via its RCL HtrA1-induced posteriorization, mesoderm induction,
and neuronal differentiation
(A-E) HtrA1 mRNA induces lack of head structures (asterisks) and secondary tail formation
(arrowhead) at stage 35. Co-injection of FLAG-PN1 mRNA restores normal head
development and blocks ectopic tail outgrowth induced by HtrA1 mRNA. FLAG-PN1pm has
weak and FLAG-PN1pm mRNA no rescuing effect.
(F-O) HtrA1 induces ectopic Xbra expression in the animal hemisphere (arrowhead in M) and
ectopic N-tubulin expression in the lateral epidermis (arrowhead in R). FLAG-PN1, but not
FLAG-PN1pm or FLAG-PN1DC, restore normal gene expression in HtrA1-injected embryos.
(P) Immunoblot analysis of embryos at stage 10.5 after co-injection of HtrA1 and FLAG-PN1,
FLAG-PN1pm or FLAG-PN1DC mRNA. α-Tubulin serves as a loading control. Note that
FLAG-PN1, FLAG-PN1pm and FLAG-PN1δC proteins were expressed at equivalent levels,
and that co-injected HtrA1 mRNA produced comparable protein amounts in each sample.
Xenopus embryos were injected into a single blastomere with 100 pg HtrA1- and 300 pg PN1-
derived mRNAs. Indicated phenotypes were: B, 21/22; C, 20/26; D, 16/26; E, 28/35; G,
53/53; H, 31/43; I, 26/46; J, 53/53; L, 55/55; M, 42/44; N, 35/41; O, 39/39. |
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Figure S6. Downregulation of each PN1 homeolog mildly stimulates mesoderm
formation and reduces anterior development
Xenopus embryos were injected with a total of 60 ng MOs into the animal pole at the 4-cell
stage. PN1.a-MO1+2 is an equal mixture of PN1.a-MO1 and PN1.a-MO2.
(A-C) Lateral view of early gastrula embryos after whole-mount in situ hybridization.
Embryos injected with PN1.a-MO1+2 (B) or PN1.b-MO (C) exhibit a slight expansion of the
mesodermal marker Xbra towards the animal pole.
(D-F) Anterior view of neurula embryos. Note the decreased expression domain of the
anterior marker Otx2 in embryos depleted of PN1.a (E) or PN1.b proteins (F).
(G-I) Anterior view of late neurulae. Embryos deficient for PN1.a (H) or PN1.b (I) show
reduced expression of the telencephalic marker Foxg1 and the posterior midbrain marker En2.
Indicated phenotypes were observed in B, 19/19; C, 14/17; E, 11/11; F, 11/12; H, 12/15; I,
13/16. |
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Figure S8. Knockdown of PN1 promotes mesoderm and suppresses ectoderm formation
in an HtrA1- and FGF-dependent manner
Embryos were separately injected with MOs and mRNAs into the animal pole at the 4-cell
stage. Hemisectioned early gastrula embryos are shown after whole-mount in situ
hybridization for Xbra (A-F) and Foxi1e (A’-F’).
(A-B’) PN1-MO, but not control-MO, induces ectopic Xbra and reduction of Foxi1e
expression in deep cells of the animal hemisphere (brackets in insets depict animal cap).
(C-D’) Injections of 200 pg HtrA1 and 2 pg Fgf4 mRNA induce ectopic Xbra at the expense
of Foxi1e expression in the inner layer of the animal cap (insets).
(E-F’) Injections of HtrA1-MO and 80 pg XFD mRNA restore normal expression of Xbra and
Foxi1e in PN1-morphant embryos.
Total injected MO amount was 60 ng (+20 ng HtrA1-MO). The frequency of the indicated
phenotypes was A, 136/144; A’, 50/59; B, 148/177; B’, 50/69; C, 21/21; C’, 47/57; D, 79/79;
D’, 93/97; E, 66/90; E’, 52/56; F, 72/83; F’, 45/51. |
