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Display additional annotations [+]
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Figure S9. PN1-MO, HtrA1, and FGF4 do not induce ectopic Papc expression in the
ectoderm of post-gastrula embryos.
Embryos were animally injected into a single blastomere at the 4-cell stage with nlacZ mRNA
as a lineage tracer (red nuclei) and processed for whole-mount in situ hybridization for Otx2
and Papc.
(A-A’’) Embryo injected with 15 ng control-MO. Strippled lines in dorsal view of neurula
embryo indicate section planes. Note Papc expression in transversally sectioned trunk
mesoderm (A’) and parasagitally sectioned tailbud mesoderm (A’’).
(B-D’’) PN1-MO (15 ng), HtrA1 mRNA (50 pg), and Fgf4 mRNA (0.5 pg) shift the Otx2-
Papc border anteriorwards (B-D), but fail to induce ectopic Papc expression in the overlying
ectoderm on the injected right side (B’-D’’).
The frequency of the indicated phenotypes was A, 79/79; A’+A’’, 4/4; B, 47/57; B’+B’’,
13/13; C, 93/97; C’+C’’, 8/8; D, 12/13; D’+D’’, 8/9. |
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Display additional annotations [+]
| Gene |
Clone |
Species |
Stages |
Anatomy |
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foxg1
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laevis
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NF stage 19
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pre-chordal neural plate
,
pre-chordal neural plate border
,
anterior neural ridge
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fgf8
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laevis
|
NF stage 24
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preplacodal ectoderm
,
posterior placodal area
,
adenohypophyseal placode
,
forebrain-midbrain boundary
|
|
szl
|
|
laevis
|
NF stage 17
|
circumblastoporal collar
,
blastopore
,
ventral mesoderm
|
|
pcdh8
|
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laevis
|
NF stage 14
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presomitic mesoderm
,
paraxial mesoderm
|
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nkx2-5
|
|
laevis
|
NF stage 20
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mesoderm
,
endoderm
,
foregut
,
cardiac mesoderm
|
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tubb2b
|
|
laevis
|
NF stage 16
|
pre-chordal neural plate
,
chordal neural plate
,
neural groove
,
neural plate
,
Rohon-Beard neuron
,
[+]
|
|
rax
|
|
laevis
|
NF stage 24
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optic vesicle
|
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en2
|
|
laevis
|
NF stage 19
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midbrain-hindbrain boundary
|
|
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Fig. 2.
PN1 promotes anterior development, suppresses mesoderm and reduces neuronal differentiation. (A) Uninjected tadpole embryo. (B) Injection of PN1 mRNA induces enlargement of head structures and coloboma (arrowhead in inset). (C) Co-injection of PN1 and HtrA1 mRNA restores normal development. (D-K) Whole-mount in situ hybridization of post-neurula embryos in anterior view. PN1 causes enlargement of the Foxg1, En2, Fgf8 and Nkx2-5 expression domains. Rax expression is not split into bilateral domains. (L-O) Xbra expression in early gastrulae, lateral view. A single marginal injection of PN1 strongly reduces Xbra expression (M). PN1 and HtrA1 partially revert this effect (N). PN1pm mRNA causes only mild or no reduction of Xbra expression (O). (P,Q) Ventral view of neurulae. PN1 expands anterior Sizzled expression. (R-X,BB) Dorsal view of neurula embryos. A single injection of PN1 causes reduction and posteriorward retraction of N-tubulin (arrowheads in S), reduction of Papc and expansion of Otx2 expression (brackets in W) on the targeted right side. PN1 and HtrA1 rescue these effects (T,BB). PN1pm does not affect these markers (U,X). (Y-AA) Injection of 15 ng HtrA1-MO, XFD mRNA or Dkk1 mRNA also causes anteriorization. (CC) Fgf4 mRNA rescues anteriorization by PN1. (DD) pCS2-Wnt3a (Wnt3a-DNA) reverts PN1-induced Otx2 expansion, but not Papc reduction. Total mRNA amounts were: PN1 constructs, 4 ng (1 ng in W,X,BB-DD, 16 ng in E,K,Q); HtrA1, 100 pg; XFD, 80 pg; Dkk1, 8 pg; Fgf4, 0.3 pg. Indicated phenotypes were shown by: B, 44/56; C, 30/30; E, 19/21; G, 16/16; I, 13/15; K, 14/17; M, 71/73; N, 18/32; O, 23/32; Q, 9/19; S, 41/42; T, 16/19; U, 42/42; W, 57/62; X, 31/39; Y, 20/24; Z, 27/31; AA, 59/60; BB, 19/23; CC, 19/24; DD, 15/16. |
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Display additional annotations [+]
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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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Figure S7. PN1 promotes anterior development via inhibition of HtrA1-, FGF- and Wnt
signals
Embryos were animally injected with MOs at the 2-cell stage and mRNAs at the 4-cell stage.
(A) Anterior view of early neurula embryos after whole-mount in situ hybridization with
Otx2, demarcating the prospective cement gland, forebrain and midbrain.
(B) PN1 mRNA (4 ng) causes expansion of Otx2 expression.
(C,D) PN1-MO (60 ng) reduces the Otx2 marker, while an equivalent amount of control-MO
has no effect.
(E,F) HtrA1-MO (20 ng) or 80 pg δXFGFR-4a mRNA restore normal Otx2 expression in
PN1 morphant embryos.
Indicated phenotypes were observed in B, 12/13; C, 59/62; D, 63/73; E, 22/24; F, 23/29. |
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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. |
