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

from dorsalized gastrulae (treated with LiCl during blastula
stages) was size fractionated and the active fraction used
to construct a plasmid cDNA library. RNAs were synthesized
from pools of plasmids and injected into ventralized
embryos produced by UV treatment (Smith and Harland,
1991). Pools of plasmid DNA that directed the synthesis
of dorsalizing RNA were sib selected until single active
clones were isolated. In the first screen we isolated Xwnf-8,
which was surprising since Xwnt-8 is down-regulated by
LiCl treatment. We therefore reexamined the initial pools
of 10,000 clones to ask whether Xwnt-8 was the only dorsalizing
activity present.
The 10 pools of 10,000 clones were analyzed by filter
hybridization for the presence of Xwnt-8. The result is
shown in Figure 1A. Xwnt-8 clones were present in the
two active pools (8 and 9), as well as in five others. The
retrospective analysis demonstrates that although Xwnr-8
is less abundant in RNA from dorsalized gastrulae, it is
still an abundant mRNA that is highly represented in the
library.
In the isolation of Xwnt-8 (Smith and Harland, 1991)
pool 9 was selected for subsequent sib selections. The
Xwnt-8 hybridization signal was weaker in pool 8 than in
pool 9 and not much different from the inactive pools that
contained Xwnf-8. To test whether pool 8 contained dorsalizing
activities other than Xwnt-8, it was subdivided into
12 pools of 1000 clones. Five of the pools had activity in
the rescue assay and three of these did not contain Xwnt-8.
The Xwnt-b-negative pool with the strongest dorsal axisrescuing
activity, pool 8.12, was further sib selected until
a single active clone was isolated (clone A3 from pool
8.12.12.A). The activity of the pools (i.e., the degree of
dorsal axis rescued) increased as progressively smaller
pools of clones were assayed. At the second sib selection
of the library (pools of 1000 clones), A3 hybridizing clones
could account for the activity of all dorsalizing pools that
A. Pool: 1 2 3 4 5 6 7 8 9 10
Activity: - - - - - - - + _ + _ -
Xwnt-6-b
Figure 1. Detection of Xwnf-8 and noggin Clones in Sib Selection
Pools
Five microgram samples of library plasmid DNA from the first (A) and
second (6) sib selections (10,000 and 1,006 clones per pool, respectively)
were digested with EcoRl and EcoRV, separated on 1% agarose
gels, and transferred to nylon membranes. The membranes were hybridized
with an Xwnt-8 probe alone (A) or first with Xwnt-8 and then
with noggin probes (6). The activities of the various pools in the dorsal
axis rescue assay are indicated (plus or minus).
did not contain Xwnt-8 (Figure 16). One pool of 1000
clones (8.2) hybridized with the A3 probe but did not have
activity in the assay (Figure 16). The reason for this is
unknown, although it is possible that this particular A3
hybridizing clone was not functional. In addition, at this
stage in the sib selection the active pools only conferred
partial rescue of dorsal development, and pools with dorsalizing
RNAs could have been missed.
Finally, preliminary results indicate that at least one additional
dorsalizing activity may be present in our library.
RNA transcribed from Ncol linearized library plasmid DNA
(rather than Notl linearized) retained axis-rescuing activity.
Ncol cleaves within the 5’ untranslated region of A3 and
within the coding region of Xwnt-8 and yields truncated,
nonfunctional transcripts. The completeness of Ncol
cleavage of A3 and Xwnt-8 plasmids in the pool was confirmed
by blotting (data not shown).
noggin cDNA Encodes a Novel Polypeptide
The 1834 nt sequence of the A3 clone is shown in Figure
2A. The sequence contains a single long open reading
frame encoding a 222 aa polypeptide with a predicted molecular
size of 26 kd. At the amino terminus, a hydrophobic
stretch of amino acids suggests that the polypeptide enters
the secretory pathway (Figure 26). There is a single
potential site for N-linked glycosylation (see the asterisk in
Figure 2A). Extensive untranslated regions are located
both 5’and 3’of the reading frame (594 and 573 bp, respectively).
The 3’ untranslated region is particularly rich in
repeated dA and dT nucleotides, and contains, in addition
to a polyadenylation signal sequence located 24 bp upstream
of the start of the poly(A) tail, a second potential
polyadenylation sequence 147 bp further upstream (both
are underlined in Figure 2A).
In vitro translation of RNA synthesized from the A3 clone
resulted in a protein product with the approximate molecular
weight predicted by the open reading frame (data not
shown). Comparison of the amino acid sequence of the
predicted polypeptide to the National Center for Biotechnology
Information BLAST network (nonredundant data
base) did not identify any similar sequence. Clone A3 thus
appears to encode a new type of protein that may be secreted
and that has dorsal-inducing activity in Xenopus.
noggin mRNA Can Rescue a Complete
Dorsal-Ventral Axis
The new sequence, and its putative protein product, were
named noggin based upon the phenotype resulting from
mRNA injection into ventralized embryos (see below). Injection
of noggin RNA into a single blastomere of a 4-cell
stage UV-ventralized embryo can restore the complete
spectrum of dorsal structures. The degree of axis rescue
was dependent upon the amount of RNA injected: the embryos
that received low doses had only posterior dorsal
structures, while embryos that received higher doses had
excess dorsal-anterior tissue. RNA transcripts from two
noggin plasmids were tested. The first (A3) contained the
full cDNA. The second @NogginAY) had a deletion removing
the first 513 nt of the 5’ untranslated region up to the
EcoRl site (see Figure 2A). The results of injection of RNA