Expression Cloning of noggin, a New Dorsalizing Factor Localized ...

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Figure 3. Dorsal Axis Rescue of Ventraked Embryos by noggin and
Xwnt-8 RNAs
Xenopus embryos were ventralized by exposure to UV light approximately
0.5 hr after fertilization. The embryos were then injected into
one blastomere at the 4-cell stage with noggin RNA transcribed either
from the full cDNA (A3) or from a plasmid containing a truncation in
the 5’ untranslated region (nogginA5’). Other embryos were injected
with Xwnt-8 RNA. The RNAs were injected at 1 to 100 pg in 10 nl of
water. Control embryos were injected with water only. Embryos were
grown until untreated embryosof the same age reached approximately
stage 41. The degree of dorsoanterior development was scored according
to the scale of Kao and Elinson (1988). The mean DAIS for 12 to
34 embryos at each RNA dose are plotted. Bars indicate the standard
errors of the mean.
noggin-Injected Blastomeres Act as a
Nieuwkoop Center
noggin, Xwnt-8, and writ-7 mRNAs all have the ability to
restore dorsal axial development when injected into ventralized
embryos (Smith and Harfand, 1991; Sokol et al.,
1991; this study). We have shown previously that when
Xwnf-8 mRNA is injected into one vegetal blastomere of
UV-treated embryos at the 32cell stage, dorsal structures
are rescued (Smith and Harland, 1991). The descendants
of the injected vegetal cells do not fate map to the rescued
dorsal tissues, but rather to the endoderm. This result is
consistent with earlier blastomere transplantation experiments
in which the strongest source of the axis-inducing
activity was found to be localized in dorsal vegetal cells
(Gimlich and Gerhart, 1984; Gimlich, 1986; Kageura, 1990).
Xwnt-8 mRNAcould also rescue dorsal development when
injected into marginal zone cells (in which case they did
contribute progeny to rescued dorsal tissues), but not
when injected into animal pole cells.
The effect of varying the site of noggin mRNA injection
was investigated in a similar manner, and the results were
similar to those observed for Xwnt-8. UV-treated embryos
at the 32-cell stage were injected with either 0.5 ng of
p-galactosidase mRNA alone or 0.5 ng of f3-galactosidase
mixed with 25 pg of nogginA5’ mRNA, as described previously
(Smith and Harland, 1991). Injection of noggin
mRNA into blastomeres of the vegetal pole (tier 4 blastomeres)
gave the most strongly dorsoanteriorized embryos
(Figure 5). Representative embryos, stained with X-gal to
indicate the fates of the injected cells, are shown in Figure
6. In both of the vegetally injected embryos the nuclear
X-gal staining was found almost exclusively in the endoderm
(the mRNA encodes a 6-galactosidase that translocates
to the nucleus, allowing distinction from the diffuse
background stain). One of the embryos shown was
strongly hyperdorsalized (DAI = -7) as a result of the
noggin mRNA injection and has a severely truncated tail
and enlarged head structures. Embryos were also rescued
by noggin mRNA injections into the marginal zone (blastomeres
from tiers 2 and 3) (Figure 5). In these embryos
6-galactosidase staining was observed primarily in the
axial and head mesoderm (Figure 6). As was observed
previously with Xwnt-8, injection of noggin mRNA into the
animal pole (tier 1 blastomeres) had very little effect on
axis formation (Figures 5 and 6). Likewise, 6-galactosidase
mRNA alone was without effect (Figure 5).
noggin mRNA Is Expressed Both Maternally
and Zygotically
In Northern blot analysis of RNA from Xenopus embryos,
two noggin mRNA species of approximate sizes 1.8 and
1.4 kb were observed (Figure 7). Figure 7A shows the
results of probing blots containing approximately 2 ug of
poly(A)’ RNA from the indicated stages with both noggin
and c-src probes (c-src serves as a control for RNA loading;
Hemmati-Brivanlou et al., 1991). A relatively low level
of noggin mRNA was detected in oocytes. By stage 11 the
level of noggin mRNA was significantly higher, reflecting
zygotic transcription (as opposed to the maternally
deposited transcripts seen in oocytes). noggin mRNA remained
at the elevated level up to the latest stage examined
(stage 45).
Based on previous results we expect the level of primary
dorsalizing RNA in our library to be elevated in LiCI-treated
embryos relative to normal or UV-treated embryos (Smith
and Harland, 1991). Figure 78 shows the relative amount
of noggin mRNA in total RNA samples from stage 8
through 10 embryos that were either untreated, UV treated
30 min after fertilization, or treated with LiCl at the 32-cell
stage. Lithium ion treatment resulted in a large increase
in the amount of noggin mRNA expressed, relative to untreated
embryos. UV treatment had the opposite effect.
noggin mRNA expression was essentially undetectable in
total RNA samples from these embryos. Thus, the abundance
of noggin mRNA in manipulated embryos parallels
the rescuing activity.
A simple model would predict that cytoplasm rotation
results in localization of dorsalizing RNA on the prospective
dorsal side of the embryo. We therefore analyzed the
distribution of noggin mRNA in oocytes and cleavage
stage embryos. Since the amount of maternally deposited
noggin RNA is too low for in situ hybridization to detect
above background, we used an RNAase protection assay.
Oocytes were dissected into animal and vegetal halves.
No enrichment of noggin mRNA was seen in either hemisphere
relative to total oocyte RNA (Figure 8). Four-cell
stage embryos were dissected into dorsal and ventral