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FIG. 1. BMP1 is ventralizing. Xenopus BMP1 mRNA was
injected radially into the marginal zone of each blastomere of
four-cell stage embryos. (A) Stage 43 tadpole following injection
of 300 pg BMP1 mRNA/blastomere. Embryos develop ectopic
structures often in the ventroposterior region and sometimes in
close apposition to the endoderm (data not shown). Embryos
injected with lower doses (30–100 pg/blastomere) also develop
these structures, but by stage 43 they are no longer morphologically
visible (data not shown). (B) Transverse section through the
posterior region shown in A (see also inset) shows an abundance
of erythrocytes. (C) A transverse section through the heart
region (inset) of an uninjected sibling showing erythrocytes for
comparison. (D–L) Whole-mount in situ hybridization analyses
comparing gene expression from uninjected control embryos (D,
G, and J) to wild-type (wt; E, H, and K) and dominant-negative
(DN; F, I, and L) BMP1-injected embryos. All embryos are vegetal
pole views oriented with dorsal toward the top. Embryos were
analyzed at early gastrula stage 10.25–10.5 following microinjection
of either 15 pg wtBMP1 or 100 pg DN-BMP1 marginally
into each blastomere at the four-cell stage. (D–F) Embryos
probed for Xsizzled. (G–I) Embryos probed for Xmyf5. (J–L)
Embryos probed for goosecoid.
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FIG. 2. Chordin can create a gradient of BMP signaling. (A)
Xvent2 is a direct target of BMP signaling. Animal caps were
dissected from late blastula stage 8.5â9 embryos and dissociated
into single-cell suspensions. Protein synthesis was inhibited by
treatment with cycloheximide (CHX) and the cells were stimulated
with purified BMP2 protein at the indicated concentrations. RTPCR
analysis for expression of Xvent2 and histone H4 mRNA is
shown. 2RT, no reverse transcriptase control. (B) 2357Xvent2/luc
differentially responds to varying amounts of BMP signaling. Increasing
concentrations of chordin mRNA were co-injected with 20
pg of 2357Xvent2/Luc reporter into a single 64-cell stage blastomere.
A semilog plot of luciferase activity at gastrula stage as a
function of amount of injected chordin mRNA is shown. (C)
Chordin can create a long-range gradient of BMP signaling. Embryos
were injected as shown in the inset box. 2357Xvent2/Luc
was injected into a single 64-cell stage blastomere, and a second
blastomere at the same stage was injected with 1 ng of chordin
mRNA. The position of the second blastomere relative to the
reporter-injected blastomere is indicated (see inset). âAâ indicates
the blastomere adjacent to the reporter-injected blastomere. Numbers
indicate blastomeres located one, two, or three cells removed
from the injected blastomere. Similar experiments were repeated
three times. The first two lanes show luciferase activity produced
in response to 2 ng of dominant-negative type I BMP receptor or 1
ng of chordin mRNAs co-injected together with the Xvent2 reporter
into the same blastomere.
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FIG. 3. Chordin’s range of action is dose dependent. (A–G and J) Chordin mRNA was microinjected together with rhodamine lysinated dextran
(RLDx) into a single animal pole blastomere at the 64-cell stage. (D–F and J) The loss of Xvent2 gene expression is shown, as detected by
whole-mount in situ hybridization (blue), in a “zone of inhibition†(white) induced by chordin. (A–C and G) Merged images of the bright-field
images in D–F and J with corresponding fluorescence images of the RLDx, which marks the progeny of the chordin-injected blastomere. Note
that, at higher concentrations of injected chordin, the zone of inhibition of Xvent2 expression expands outside of the RLDx fluorescing “cloneâ€
of cells. At the highest dose of chordin shown (300 pg; A, D), the boundary (which is not sharp) between the Xvent2-expressing cells and the zone
of inhibition of Xvent2 is approximately 450 mm from the edge of the chordin-expressing fluorescent cell population (yellow arrow). (H, I, K, L)
Dominant-negative type I BMP receptor (2 ng) and “cleavage mutant†BMP4 (2 ng) do not act at a distance and behave like low concentrations
of chordin in this assay. The asterisk in C marks the narrow area around the chordin-expressing cells which are not expressing Xvent2. (M–O)
Xvent2 expression can be rescued by elevating the level of expression of BMP1 in cells within the zone of inhibition of BMP signaling induced
by chordin. The arrow in N points to a region of rescued Xvent2 expression overlapping a BMP1-expressing clone of cells (see O). All embryos
in A–L, N, andOare animal pole views. (P) Levels of chordin corresponding to chordin’s short-range action are sufficient to induce secondary axes.
Tadpole stage embryos showing moderate-sized secondary axes (arrows) induced by injection of 3 pg of chordin mRNA into a single ventral
marginal zone blastomere at the 64-cell stage. At this dose 57% of injected embryos (n 5 30) developed secondary axes and 10 pg of chordin
induced secondary axes in 75% of injected embryos (n 5 28).
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FIG. 4. Dominant-negative BMP1 inhibits wild-type BMP1âs activity in vitro and in vivo. (A) Construction of a dominant-negative BMP1
protein (DN-BMP1). The C-terminal three CUB repeats and one EGF repeat of BMP1 were fused in frame with the prodomain of Xenopus
activinbB, which had its ligand domain discarded. The DN-BMP1 protein is expected to be processed at the activin maturation cleavage site
(inverted triangle) upstream of a single FLAG epitope tag located between the cleavage site and the first CUB repeat. (B) Western
immunoblotting of transfected COS cell supernatants using anti-FLAG antibody shows that DN-BMP1 is synthesized, secreted, and
processed. Lane 1, control untransfected cell supernatant. Lane 2, a band migrating at approximately 60 kDa corresponding to FLAG-tagged
processed DN-BMP1. Lane 3, the reduced mobility of DN-BMP1 in lane 2 is likely due to its glycosylation as treatment of transfected cells
with the glycosylation inhibitor tunicamycin increases the migration of DN-BMP1 to approximately 52 kDa. (C) DN-BMP1 inhibits
chordin proteolysis by wild-type BMP1 in vitro. Transfected 293T cell supernatants were incubated with chordin protein tagged at its
C-terminus with a myc epitope (;120 kDa; open arrowhead). Lane 1, control pCS21-transfected supernatants do not contain significant
chordin-cleaving activity. Lane 2, supernatant containing wild-type (WT) BMP1 leads to a decrease in the amount of full-length chordin
protein and the appearance of faster migrating (;95 kDa; closed arrowhead) cleavage product. Lane 3, supernatant containing DN-BMP1 has
no chordin-cleaving activity. Lane 4, DN-BMP1, when cotransfected together with wild-type BMP1, blocks chordin cleavage. (DâH)
DN-BMP1 inhibits the activity of wild-type BMP1 and wild-type Xolloid in vivo. (E, G) Expansion of the ventral blood island is observed
following microinjection of 15 pg of wild-type BMP1 or wild-type Xolloid marginally into each blastomere at the 4-cell stage (compare to
uninjected sibling control in D). (F, H) Injection of 50â150 pg of DN-BMP1 with 15 pg wild-type BMP1 or wild-type Xolloid abrogates the
ventral blood island expansion phenotype.
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FIG. 5. Dominant-negative BMP1 is dorsalizing. (A) One hundred picograms of DN-BMP1 was injected into the marginal zone of a ventral
vegetal blastomere at the 8-cell stage. Most embryos (89%, n 5 57) develop secondary axes which were incomplete as they lack anterior
structures; however, a small percentage (;3.5%) were cyclopic. Different batches of embryos respond differently to DN-BMP1 (data not
shown). In many batches, 100 pg is insufficient to induce secondary axes, but 300 pg will induce secondary axes efficiently. Usually, 1 ng
of DN-BMP1 induces gastrulation defects. At these higher doses of DN-BMP1, the appearance of cyclopic heads and heads containing two eyes rises to approximately 30% of injected embryos. (B) Co-injection of 30 pg lacZ mRNA together with 100 pg DN-BMP1 into a single
ventral vegetal blastomere at the 8-cell stage results in strong staining in the secondary axes of tailbud stage embryos. This result shows
that DN-BMP1 functions in the region where it is expressed, converting ventral marginal zone to dorsal organizer tissue, and does not need
to act at a distance to induce secondary axes. (C) Microinjection of 100 pg of DN-BMP1 into the marginal zone of both ventral blastomeres
at the 4-cell stage induces secondary axes (arrows), and formation of these axes can be blocked (D) by co-injection with 60â100 pg of
wild-type BMP1 or Xolloid mRNA (data not shown).
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FIG. 6. Axis rescue by dominant-negative BMP1 suggests that chordin expressed outside of Spemannâs organizer may be negatively
regulated by BMP1 and Xolloid. (A) Hypothesis 1: Chordin protein, synthesized by cells in Spemannâs organizer (left, dark blue), may act
at a distance (e.g., by diffusing away from the organizer; arrows). Normally, this chordin protein may be the target of degradation by
endogenous BMP1 and Xolloid. DN-BMP1 blocks chordinâs degradation leading to development of secondary axes. (B) Hypothesis 2:
Chordin protein, while synthesized by cells in Spemannâs organizer (left, dark blue), may also be distributed at a low level throughout the
embryo (left, light blue), perhaps as maternally expressed mRNA or protein or as low-level uniform zygotic expression. DN-BMP1
expression on the ventral side may block BMP1 and Xolloid thereby allowing this uniformly expressed chordin protein to induce
development of secondary axes. Embryos depicted to the right in A and B show the effects of UV treatment during the first cell cycle, which
blocks expression of chordin in the organizer at gastrula stage (Sasai et al., 1994, and data not shown), not nonorganizer chordin expression.
Therefore, assessing DN-BMP19s ability to induce an axis in UV-hyperventralized embryos permits determination of whether organizer
expression of chordin is necessary for the axis-inducing activity of DN-BMP1 (CâF). (C) UV-irradiated embryos which are hyperventralized
(DAI 5 0.13, n 5 46). (D) UV-treated embryos injected marginally at the 8-cell stage with 100 pg of DN-BMP1 mRNA (DAI 5 3, n 5 27).
(E) Axis rescue was blocked by co-injection of either 60 pg wild-type (WT) BMP1 or 60 pg wild-type Xolloid (data not shown) mRNAs
together with 100 pg DN-BMP1. Therefore, we conclude that DN-BMP1 functions to induce axes by blocking endogenous BMP1 and
Xolloid. (F) Sibling control embryos used for staging. (GL) Axis rescue by DN-BMP1 in embryos hyperventralized by dominant-negative
TCF3 (DN-TCF3). It has previously been shown (Laurent et al., 1997; Medina et al., 1997) that inhibition of organizer formation by UV
treatment during the first cell cycle results in ectopic expression of some organizer marker genes in the vegetal pole. We wished to rule out
the possibility that axis rescue by DN-BMP1 could be due to a modified version of hypothesis 1, in which chordin is instead expressed in
the vegetal pole and âdiffusesâ into the marginal zone. Similar to hypothesis 1 above, degradation of this chordin by BMP1 and Xolloid may
be inhibited by DN-BMP1. To rule out this possibility, we sought to rescue axis formation using DN-BMP1 following inhibition of
organizer formation by DN-TCF3. (G) Hyperventralized embryos following injection of 250 pg marginally into the dorsal side of each
blastomere at the 2-cell stage. lacZ mRNA was co-injected together with DN-TCF3 to mark tissue derived from the injected side. (H) Axes
in embryos injected dorsally with DN-TCF3 and lacZ were partially rescued by ventral injection at the 8-cell stage with 100 pg of DN-BMP1
into each of the two vegetal blastomeres. Note that the lacZ staining, marking cells which would have been dorsal, now marks ventral cells,
while axes are derived from unmarked ventral tissue. (I) Control embryos injected in the dorsal marginal zone with lacZ mRNA alone
showing the normal distribution of lacZ into dorsal structures. (J) Control embryos injected in the ventral marginal zone with lacZ mRNA
alone showing the normal distribution of lacZ into ventroposterior structures. (K) DN-TCF3 inhibits chordin expression in the organizer.
(L) A sibling embryo showing chordin expression in the organizer. K and L are vegetal views. Dorsal is toward the top in L.
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FIG. 7. Chordin is expressed outside of Spemannâs organizer and
BMP1 acts to inhibit a chordin positive autofeedback loop. (A)
Chordin is expressed outside of Spemannâs organizer. Marginal
zones fragments were dissected from early gastrula stage 10.25
embryos (numbers on the embryo correspond to RT-PCR lane
numbers) and chordin RNA was examined by RT-PCR. While
chordin is expressed at very high levels dorsally, it is detectable in
the ventral marginal zone as well. (BâE) Chordin gene expression is
up-regulated by repressing BMP signaling. Ventral expression of
chordin itself (lacking its 39UTR; 20 pg), dominant-negative type I
BMP receptor (1.6 ng), or noggin (10 pg) results in ventral expression
of chordin mRNA. (E) Chordin is also induced in the animal pole in
response to inhibition of BMP signaling (arrow). Noggin mRNA (10
pg) was co-injected animally at the 64-cell stage. (F) Chordin is also
up-regulated ventrally and in the animal pole (G) upon expression
of DN-BMP1. In BâG, the mRNA injected is stated in the lower
right corner. In B, chordinâs 39UTR was used as probe to selectively
detect mRNA synthesized off the endogenous chordin gene.
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FIG. 8. Competition between BMP4 and chordin positive autofeedback loops. BMP4 signaling (left) through heteromeric type I and II BMP
receptor complexes results in the concomitant repression of chordin gene expression and activation of BMP4 gene expression, thereby
maintaining expression of BMP4 outside the Spemannâs organizer. A chordin positive autofeedback loop (right) occurs indirectly through
chordinâs repression of the BMP4 positive autofeedback loop. Chordin, by competitively inhibiting BMP4 receptor activation, blocks BMP4
signaling-dependent activation of its own gene expression and, concomitantly, chordin blocks BMP49s repression of expression of the
chordin gene. The chordin gene is then derepressed, completing the loop. The balance between these two feedback pathways depends on
the levels of expression of BMP4 (ventrally, laterally, and animally) and chordin (dorsally; Spemannâs organizer). These are established at
midblastula transition by the dorsal determinant pathway, in the case of chordin, and the âdefaultâ ventral state of the remainder of the
embryo, also established by maternal factors. Low level expression of chordin protein outside of the organizer is inhibited by BMP1 and
Xolloid which proteolytically inactivate chordin, thereby permitting maintenance of the ventral (BMP4 autofeedback) pathway. BMP signal
transduction resulting in activation and repression of the BMP4 and chordin genes may or may not occur by direct pathways.
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