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

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lower than the quantity needed for axis rescue of ventralized
embryos by injection. However, translational or
posttranslational control might normally enhance the activity
of noggin protein on the dorsal side, so large amounts
of transcript may need to be injected into ventralized embryos
to overcome reduced activity.
To test whether noggin is required for normal development,
its expression or activity must be eliminated experimentally.
We have demonstrated a correlation between
ventralization of the embryo and elimination of zygotic noggin
expression, but the amount of maternal mRNA is unaffected
in ventralized embryos, and noggin mRNA is present
in the ventral half of the cleaving embryo. Whether
protein expression or activity is affected in ventral cells is
not yet known. In principle, maternal noggin mRNA can be
eliminated by oligonucleotide-mediated RNAase H digestion
and the effects on development monitored (Kloc et al.,
1989). Our recent cloning of the mouse homolog of noggin
(A. Huang, W. C. S., and R. M. H., unpublished data) will
also facilitate genetic studies.
Dorsal Mesodermal Patterning in Xenopus
To date, mesoderm induction assays and axis-rescuing
assays have identified different molecules. The mesoderm
inducers include activin and fibroblast growth factor (FGF).
Injection of FGF RNA has little effect on normal embryos
(Kimelman and Maas, 1992) and activin mRNA injection
produces only partial axes (Thomsen et al., 1990; Sokol et
al., 1991). Although a concentration gradient of mesoderm
inducer could in principle account for dorsal-ventral differences
(Green and Smith, 1990), the discovery of distinct
axis-rescuing (dorsalizing) molecules suggests a different
mechanism for patterning the mesoderm. While molecules
like Xwnt-8 have no inherent mesoderm-inducing
activity (Christian et al., 1992) they sensitize cells to mesoderm
inducers; thus, the combination of a ventral mesoderm
inducer, FGF, with cells expressing Xwnf-8 results
synergistically in differentiation of dorsal mesoderm (Christian
et al., 1992). In preliminary experiments, we observed
that animal caps exposed to noggin formed very little, or
no, mesoderm on their own. However, when combined
with a low level of activin, the animal caps differentiated
a large amount of dorsal mesoderm (A. Knecht and
W. C. S., unpublished data). Therefore, wnt mRNAs and
noggin appear to have similar properties as dorsalizing
factors that sensitize cells to mesoderm inducers.
A requirement for the dorsalizing class of molecules in
the embryo is also suggested by the observation that ventral
animal cap tissue exposed to high concentrations of
activin cannot form notochord or anterior neural tissues
(Sokol and Melton, 1991; Bolce et al., 1992); in contrast,
dorsally derived cells can respond to activin to form these
structures. In the embryo the pattern of the mesoderm
could be generated by a uniform distribution of mesoderm
inducers in the marginal zone that is acted on by a local
source of dorsalizing factor such as a wnt or noggin (reviewed
by Kimelman et al., 1992).
Experimental
Procedures
Production of Xenopus Embryos
Xenopus embryos were prepared as described previously (Condie and
Harland, 1967). Embryos were staged according to the table of Nieuwkoop
and Faber (1967). Ventralized embryos were produced by UV
irradiation with a Stratalinker (Stratagene), and dorsalized embryos
were produced by treatment with LiCl (Smith and Harland, 1991).
Isolation and Sequencing of noggin cDNA
The construction of the size-selected plasmid cDNA library from stage
11 LiCI-treated embryos has been described previously (Smith and
Harland, 1991). In vitro RNA synthesis, injection assay for dorsal axis
rescue, and sib selections were also done as described previously
(Smith and Harland, 1991). A slightly different protocol was used in
plating bacteria for preparation of template plasmid DNAs from the
sib-selected pools. As before, agarose plate cultures were used to
prepare plasmid DNAs from pools of 100,000 to 1,000 clones per pool.
This was done in order to ensure that particular clones did not become
greatly overrepresented, as would be more likely in liquid cultures.
However, for pools of 100 clones agarose plates with 100 colonies
were first grown overnight. Replica plates with ten impressions from
the original plates were then made, grown overnight, and used to
prepare template DNA. Pools of ten were produced first by picking and
growing small liquid cultures of the 100 individual colonies from the
original plate of the active pool of 100 clones. These cultures were then
pooled into ten pools of ten clones, while reserving some of each single
clone for later expansion and assay.
To test for the presence of Xwnf-8 or noggin sequences in library
pools, 5 Kg of DNA from each pool was digested with EcoRl and
EcoRV, which released the cDNA inserts from the plasmid vector.
Blots made from the samples after electrophoresis on a 1% agarose
gel were hybridized with Xwnt-8 and noggin probes.
The nucleotide sequence of both strands of the isolated noggin
cDNA clone was determined by the dideoxy termination method
(Sanger et al., 1977) using modifiedT7 DNApolymerase(US Biochemical
Company). Deletions were prepared in sequencing templates by
both restriction enzyme and exonuclease Ill digestion (Henikoff, 1967).
RNA Isolation and Analysis
Total RNA was isolated from embryos and oocytes by a small-scale
protocol described previously (Condie and Harland, 1967). Oocytes
and blastula stage embryos were fixed in 96% ethanol plus 2% acetic
acid prior to dissection. The dissected tissues were then rinsed twice
in 100% ethanol before homogenization.
Samples containing either the total RNA equivalent of 2.5 embryos
or approximately 2 ug of poly(A)’ RNA were analyzed by Northern
blotting as described previously (Smith and Harland, 1991; Condie and
Harland, 1967). Random primed DNA probes were prepared from a
1323 bp fragment of noggin cDNA from the EcoRl site at nucleotide
-63 to an EcoRV site that lies in the vector immediately 3’ to the end
of the cDNA. Other probes used in the present study (Xenopus c-sfc
and EFla) have been described previously (Hemmati-Brivanlou et al.,
1991; Krieg et al., 1969).
RNAase protection assays were done using a protocol detailed previously
(Melton et al., 1964) with minor modifications (C. Kintner, Salk
Institute, La Jolla, CA). A noggin cDNA exonuclease Ill deletion clone
(clone 5.5, see Figure 2A) having a deletion from the S’end to nucleofide
363 was used as a template for synthesizing RNA probes. The
template DNA was linearized by EcoRl restriction enzyme digestion,
and a 463 base antisense RNA incorporating =P was synthesized with
T7 RNA polymerase. A 367 base antisense EFia RNA probe was used
as a control for amount of RNA per sample (Krieg et al., 1969). Probes
were gel purified prior to use.
In Situ Hybridization
The procedure described by Frank and Harland (1991) and detailed in
Harland (1991) was used with minor modifications. After fixation and
storage, the embryos were checked to ensure that the blastocoel and
archenteron were punctured. Care was taken to puncture the residual