|
FIG. 1. Inhibition of Nodal signaling does not prevent CNS formation. (AâD) External and histological views of embryos injected radially
into the vegetal pole of each blastomere at the 4-cell stage with 150 pg cer-S (n 5 167), 1.5 ng tAlk4 (n 5 21), or 1.5 ng antivin mRNA (n 5
89) at stage 32. The cyclopic eyes are indicated by arrowheads. (E) RT-PCR analysis of the same embryos showing expression of NCAM,
but a decrease of the mesodermal markers a-actin and a-globin caused by the three anti-mesodermal agents. EF1-a serves as a loading
control. (FâJ) Whole-mount in situ hybridization analyses of control, cer-S-, and DN-XTcf-3-injected embryos with the neural plate marker
Sox-2 at stage 12.5 (FâH, dorsal view) and BMP-4 at stage 10.5 (I and J, lateral view). D, dorsal; V, ventral.
|
|
FIG. 2. Neural induction is dependent on cortical rotation. (A) RT-PCR analysis of embryos that have been irradiated with UV light (lanes
3â6) and injected radially in the marginal zone at the 4-cell stage with cer-S mRNA (lanes 2 and 4), cer-S and b-catenin (150 pg each) mRNA
(lane 5), or cer-S and tBR (1.5 ng) mRNA (lane 6). Note that expression of the neural markers NCAM, Otx-2, Krox-20, and En-2 is inhibited
by UV irradiation, but is restored after injection of either b-catenin or a dominant-negative BMP receptor (three embryos per sample; three
independent analyses). (B, C) Histological analysis of embryos injected with cer-S mRNA at stage 42. UV treatment results in the loss of
neural structures in cer-S-injected embryos (n 5 81). fb, forebrain; hb, hindbrain.
|
|
FIG. 3. Preorganizer expression of chordin requires the early b-Catenin pathway for its initiation, and Xnrs for its maintenance in the
gastrula organizer. Whole-mount in situ hybridizations with chordin probe of embryos injected with cer-S (600 pg), DN-XTcf-3 (600 pg), or
uninjected controls at stages 8 (AâC), 9 (DâF), 10 (GâI), and 10.5 (JâL). A patch of chordin expression is detectable at least 2 h before the
external dorsal lip is seen at stage 10 (three independent experiments). All embryos are shown in dorsal view; pigmented embryos with
strong dorsalâventral polarity were used in these experiments.
|
|
FIG. 4. Expression of organizer marker genes at the blastula and gastrula. (A, B) In situ hybridization on paraffin sections with a chordin
probe at stages 9 and 10.5. Embryos were sectioned sagittally along the dorsalâventral axis. Note the broad expression domain at stage 9,
resembling the area of nuclear localization of b-Catenin (Schneider et al., 1996). (C) RT-PCR analysis of embryos at stages 9 and 10.5 in the
presence or absence of microinjected cer-S mRNA. Many classical organizer genes can be detected as early as stage 9 (lane 1) and chordin,
noggin, follistatin, and cerberus continue to be expressed in the absence of Xnr signaling (lane 2). At stage 10.5, all organizer markers, as
well as Xbra, are inhibited by the anti-Xnr reagent Cer-S (lanes 3 and 4). The lack of Xbra expression at stage 9 indicates that, at this early
blastula stage, mesoderm induction has not yet taken place. (D) RT-PCR analysis of embryos injected with DN-XTcf-3 (lane 2) and untreated
controls (lane 1) at stage 9, showing that the induction of many organizer genes is dependent on an early b-Catenin signal. ODC serves as
an RNA loading control.
|
|
FIG. 5. The b-Catenin pathway is sufficient to induce preorganizer gene expression program at the blastula stage. (AâD) Embryos were
injected into the animal pole at 4-cell stage with synthetic mRNA for b-catenin (150 pg per blastomere), dnGSK-3 (150 pg per blastomere),
or were treated with LiCl and analyzed at stage 9 for chordin expression by in situ hybridization. The patch of expression of chordin in (A)
marks the position of the preorganizer on the dorsal margin of blastula embryos. All embryos are shown in animal view. (E, F) Ectodermal
explants of the embryos treated the same way as above were isolated at stage 8 and the expression levels of chordin, noggin, follistatin, and
Xnr3 (E) or cerberus, dkk-1, frzb-1, and gsc (F) were determined by RT-PCR at stage 9. EF1a and ODC serve as control for equal loading.
Note that organizer gene markers in (E), but not those in (F), were induced by the b-Catenin pathway in ectodermal explants at blastula.
|
|
FIG. 6. b-Catenin signaling promotes neural differentiation and neural crest formation. (A) RT-PCR analysis of ectodermal explants
injected with dnGSK3 (lane 3; 150 pg/blastomere), b-catenin (lane 4; 150 pg/blastomere), treated with LiCl (lane 5; 120 mM in 0.13 Barth
for 30 min at 32-cell stage), or injected with a constitutive active BMP receptor (caBR; 1 ng/blastomere) and treated with LiCl (lane 6) and
harvested at stage 24. Note that induction of the neural marker genes NCAM and Otx-2 is activated by the b-Catenin pathway and requires
inhibition of BMP signaling. (B) Ectodermal explants of embryos treated with LiCl express the neural crest marker Slug by RT-PCR analysis.
(CâK) Ectodermal explants microinjected with dnGSK3 or b2catenin mRNA and analyzed by in situ hybridization using b-neurotubulin
as a marker for differentiated neurons at stage 24 (CâE) or by morphological (FâH) and histological (IâK) criteria at stage 42. The inset in (C)
shows the expression of b-neurotubulin in a control embryo. Note that, in explants of dnGSK3 and b-catenin-injected embryos,
b-neurotubulin expression is patchy and that, in histological sections, neural crest-like tissues with melanocytes are observed. ae, atypical
epidermis; ne, neural tissue; nc, neural crest; cg, cement gland.
|
|
FIG. 7. Model for organizer induction. The diagram indicates
three steps in the establishment of a dorsal signaling center. At the
blastula stage, nuclear b-Catenin (dotted area) induces the early
zygotic expression of organizer-specific genes (black), such as
chordin, noggin, follistatin, and Xnr3 in the preorganizer region.
These BMP antagonists may participate in the predetermination of
the neural plate. Later, Nodal signals originating from vegetal cells
(hatched area) are required for the induction of mesoderm and for
maintenance of organizer gene expression. At the gastrula stage,
the same cocktail of factors secreted by the mature Spemannâs
organizer will pattern all three germ layers and is maintained by
Nodal-related signals produced from within the mesoderm
(hatched area).
|
