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Fig. 1. Expression of dorsoanterior mesoderm markers in nodalinjected
embryos. (A) goosecoid transcript levels are elevated during
early gastrula stages and remain higher than controls at least into
sibling neurula stages. ODC signal assesses RNA loading in the
samples. (B) Muscle-specific (ms) actin expression is upregulated in
nodal-injected embryos. Higher levels of ms-actin mRNA are
detected precociously in embryos expressing nodal. Cytoskeletal
actin serves as a control for RNA integrity in the lanes. C, control; N,
mouse nodal RNA-injected.
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Fig. 2. Characterization of
animal caps induced by
injection of mouse nodal
RNA. Morphological (A,B),
and histological (C,D)
analysis of control and
nodal-loaded animal caps.
(A,C) Control caps form
atypical epidermis, but caps
expressing mouse nodal
(B,D) elongate and
differentiate dorsal
mesodermal tissues, often
consisting almost entirely of
notochord and small patches
of muscle. Abbreviations:
epi, atypical epidermis; ms,
muscle; no, notochord.
(E) Analysis of gene
expression induced in
animal explants by different
nodal RNA doses. High
doses (1 ng RNA/embryo)
induce goosecoid (a
dorsoanterior mesoderm
marker), muscle-specific actin and Xbra. Decreased doses (100, 10, or 1 pg/embryo) do not induce goosecoid, but intermediate concentrations
induce muscle-specific actin and Xbra. All samples are from the same injection experiment. goosecoid and Xbra were assayed at sibling stage
10. 5, and muscle actin at stage 20. ODC and cytoskeletal actin serve as controls for RNA integrity and loading in samples.
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Fig. 3. Alignment of amino acid sequences of mouse nodal with Xnr-1, Xnr-2 and
a chicken nodal-related peptide. (A) Deduced amino acid sequences of Xnr-1 and
Xnr-2. A hydrophobic region (underlined) at the N terminus of each Xnr
resembles a secretory signal sequence, with cleavage predicted according to the
algorithm of von Heijne (1986). Four potential N-linked glycosylation sites
(consensus N-X-T/S) are present in each protein (centered on residues 72, 137,
174, and 345 for Xnr-1, and residues 72, 160, 174, and 344 for Xnr-2). Three are
positionally conserved between Xnr-1/Xnr-2. Putative basic proteolytic
processing sites (RRxRR, underlined) begin at residues 277 (Xnr-1) and 278
(Xnr-2). Asterisks indicate identities, double dots represent conservative changes.
(B) Alignment of C-terminal mature regions of Xnr-1, Xnr-2, mouse nodal and a
newly isolated chick nodal-related sequence. Alignments begin at the putative
basic processing site of each molecule. The region of cysteine spacing unique to
the Xnr factors (C-X-X-C) is underlined. Vertical lines represent identities in all
four proteins. A consensus sequence is presented below the alignment. Dashes
represent spaces introduced to optimize alignments.
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Fig. 4. Temporal and spatial expression of Xnr-1 and Xnr-2 during
Xenopus development. (A) RNAse protection analysis of Xnr-1 and
Xnr-2 expression during Xenopus development. Transcripts are detected
during late blastula (stage 9) and gastrula (stage 10 and 10. 5) stages. A
very low level of Xnr-1 RNA is detected during neurula stages (stage
17), but no expression is detected for Xnr-2 after gastrulation (stage
13). Stage 8 represents an RNA pool before zygotic transcription
begins, and transcripts for Xnr-1 and Xnr-2 are not detected. ODC is a
loading control, and the tRNA lane demonstrates specificity of signal to
embryo RNA. (B-H) Whole-mount in situ hybridization analysis of Xnr-1 and Xnr-2 expression. All embryos are cleared albino embryos,
viewed from the vegetal surface with dorsal oriented upward. The dorsal lip is indicated by the black arrowhead. (B) Stage 9 embryos show
punctate perinuclear Xnr-1 signal over the entire vegetal region. Xnr-2 shows the same pattern (data not shown). (C) Xnr-1 signal at stage 10.25
is restricted to the dorsal marginal zone (dark arc at bottom left is a background artefact). (D) Xnr-2 signal in stage 10 pregastrula is primarily
located in the dorsal marginal zone, but also in adjacent dorsovegetal cells. (E) Xnr-2 signal in the stage 10.5 gastrula is highly concentrated
just above the dorsal lip, with a gradual decrease laterally and ventrally. (F) Whole-mount stained stage 10.25 embryo, split open along the
dorsal/ventral plane and viewed internally to show Xnr-2 expression at the dorsal lip. Superficial and slightly deeper staining is observed. Some
out-of-focus vegetal cells below the lip express Xnr-2 (white arrowhead). (G) noggin mRNA hybridization in a stage 10.5 embryo shows
deeper mesodermal expression extending anteriorly along the dorsal midline. (H) Xnr-2 sense strand control, stage 10.25 embryo. (I) RNAse
protection analysis of Xnr-1 and Xnr-2 distribution in dissected gastrulae. Lanes 1-3: at stage 10.25, Xnr-1 and Xnr-2 transcripts are detected in
the marginal zone, at greatly reduced levels in vegetal tissue, but are undetectable in animal tissue. Lanes 4 and 5: in stage 10 embryos, Xnr-1
RNA and, to a lesser extent, Xnr-2 RNA, is enriched in dorsal halves of embryos compared to ventral halves. EF1-a assesses RNA integrity
and loading.
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Fig. 5. RNAse protection analysis of Xnr-1 and Xnr-2 expression in
animal caps treated with growth factors and LiCl- and UV-treated
embryos. (A) Xnr-1 and Xnr-2 expression is induced in animal caps
by activin, but not FGF, indicating activation by dorsal mesoderminducing
signals. Control protections show Xnr-1 and Xnr-2 RNA in
stage 10.5 sibling embryos. EF1-a assesses RNA loading in the
samples. (B) Expression of Xnr-1 and Xnr-2 at stage 10 in UVventralized
or LiCl-dorsalized embryos. UV-ventralization greatly
reduces Xnr-1 and Xnr-2 transcript levels. In contrast, LiCl
dorsalization results in a 2-4 fold increase (evaluated
densitometrically) in Xnr-1 and Xnr-2 RNA compared to untreated
siblings. EF1-a is used as a loading control. DAI, dorso-anterior
index score of sibling embryos (Kao and Elinson, 1988).
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Fig. 6. Effects of Xnr-1 and Xnr-2 on animal cap
explants. Morphological (A-C) and corresponding
histological analysis (D-F) of caps explanted from
Xnr-1 or Xnr-2 RNA-injected embryos. (A,D) Control
explants remain rounded, differentiating into atypical
epidermis. (B,E) Most Xnr-1-injected caps extend
slightly compared to controls, and primarily
differentiate blocks of striated muscle (ms).
Notochord differentiation is observed in Xnr-1
injected caps, but at lower frequency than Xnr-2.
(C,F) Xnr-2 expressing explants elongate extensively,
and consistently form dorsal mesodermal tissue
including notochord (no) and striated muscle.
(G) Induction of dorsal mesodermal markers in animal
caps by Xnr-2 mRNA. Explants from embryos
injected with high Xnr-2 RNA doses (100 pg)
differentiate dorsoanterior mesoderm as marked by
goosecoid and muscle actin expression. Lower concentrations do not induce goosecoid, but still induce
actin and the pan-mesodermal marker, Xbra. As little as 1 pg of Xnr-2 RNA injected into 1-cell embryos
induces actin and Xbra expression in animal caps (barely visible in this exposure). goosecoid and Xbra
were assayed at sibling stage 10.5, and actin at sibling stage 20. Sibling embryo RNAs provide positive
controls, and EF1-a and cytoskeletal actin assess RNA integrity and loading. (H) While high Xnr-2
RNA doses induce dorsal mesoderm markers (compare to G), intermediate doses of Xnr-2 RNA induce
the ventrolateral mesodermal markers Xhox-3 and globin. The highest levels are seen at 10 pg/embryo.
Low levels of Xhox-3 are indicated by the arrowheads.
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Fig. 7. Muscle-specific actin expression in ventral marginal zone explants dorsalized by Xnr-1
and Xnr-2 expressed zygotically. (A) Ventral marginal zones (VMZs) isolated from stage 10
gastrulae previously injected at the 4-cell stage with pCSKA:Xnr-1 or pCSKA:Xnr-2 express
muscle-specific actin (ms-actin), indicating dorsalization compared to control VMZ explants,
which do not express ms-actin. In four separate experiments (two separate examples are shown),
pCSKA:activin never induced detectable levels of ms-actin. Dorsal marginal zone (DMZ)
explants express high levels of msactin.
(B-E) Histological analysis
of explanted marginal zones.
(B) Control VMZs differentiate
ventral-type tissues, including
mesothelium (mt) and loose
mesenchyme. ym, yolk mass.
(C) Control DMZs form notochord
(no), striated muscle (ms), and
neural tissue (nt). (D,E) VMZ
explants preinjected with
pCSKA:Xnr-1 or pCSKA:Xnr-2,
respectively, are dorsalized and
differentiate large blocks of striated
muscle.
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Fig. 8. Complete axial rescue of UV-ventralized embryos by
localized Xnr-1 RNA injection. (A) Normal tadpole, (B) UV-treated
embryo (DAI=0; Kao and Elinson, 1988) at the same age of
development, (C) Embryo resulting from injection of Xnr-1 RNA
into one cell of a 4-cell stage UV-irradiated embryo. Except for a
small injection artefact in the belly, Xnr-1 rescued embryos are
indistinguishable from sibling normal embryos. In a representative
experiment, 13 of 21 injected embryos (61%) were rescued to a DAI
of 3-5, and 8 of these 13 had a DAI of 4 or 5, representing complete
rescue. A DAI score of 3 was recorded if definitive melanized eye
tissue was seen, while a DAI of 5 represents a normal tadpole.
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