Fig. 1. Structure and expression of XBMPRII. (A) Functional
domains of XBMPRII are defined by percentage amino acid
similarity to human BMPRII (Kawabata et al., 1995). The dominant
negative XBMPRII (tBRII) is truncated 10 amino acids after the
putative transmembrane domain (arrow). PM, plasma membrane.
(B) RNAse protection analysis during development (stages indicated
above the lanes) shows maternal and zygotic XBMPRII transcription.
RT-PCR detection of the constitutive fibroblast growth factor
receptor (FGFR) RNA was used as a loading control.
Fig. 2. Spatiotemporal expression pattern of XBMPRII by whole-mount in situ
hybridization. (A) Side view of a stage 9 blastula showing enhanced signal in
the animal region. (B) Sense probe control – neurula stage. (C) The pattern in A
was confirmed by RNAse protection analysis of XBMPRII in animal cap (AC)
and vegetal pole (VP) explants from stage 9 embryos. EF1a was used as a
loading control (four AC or ten VP explants/lane). (D) Vegetal and dorsovegetal
views of stage 10 (left) and 10.5 (right) embryos show expression restricted to
the marginal zone, with substantially less expression adjacent to the forming
dorsal lip (arrowheads). (E) Eosin-counterstained sections of stage 10.5 embryos
show expression in preinvoluting mesodermal precursors (bracketed), but not
extending to the dorsal lip margin (arrowhead). (G) At the neurula stage,
expression is in the posterior, preinvoluting mesoderm. (H) Sectional analysis at
this stage (plane indicated by bars in G) demonstrates a ring of signal
surrounding the yolk plug (YP), and at higher magnification (L – area boxed in
H), the subepithelial localization of expression. (J) At late neurula stages,
expression is concentrated in two areas of ventral mesoderm with fainter
expression in the brain and eye (e). (K) Sections through the posterior focus
(bars in J indicate plane) demonstrate subepithelially localized expression
extending from the proctodeum (PR) to midway up the lateral wall (bars
indicate dorsal limit). Faint neural tube staining is seen (arrowhead). (N) and (O)
A complex pattern of expression is seen at the tailbud stage, including
expression in neural tube (NT), brain (BR) and around the otic vesicle (OV).
CG, cement gland. F, I, M and P show BMP4 expression at similar stages
(photographs kindly provided by E. M. De Robertis), demonstrating a high
degree of overlap with XBMPRII expression. In B,G,I,J and M-P, anterior is to
the left, and dorsal uppermost. Arrowheads in F and P indicate dorsal lip and
ventral blood islands, respectively.
Fig. 3. Secondary axis induction by ventral tBRII injection.
(A) Wild-type and (B) tBRII-injected embryos at the neurula stage.
The arrowhead indicates the secondary axis, which lacks a visible
floorplate. (C) Wild-type embryo at the tadpole stage. (D) Embryo
injected ventrally with tBRII exhibits a secondary axis (arrows)
lacking normal head morphology. (E) H&E-stained section of a
tadpole stage tBRII RNA-injected, secondary axis embryo. The
endogenous axis contains organized somites (SO), neural tube (NT),
notochord (NO) and gut (G), while the ectopic axis (boxed area)
comprises highly disorganized tissues. (F) Higher magnification of
area boxed in E. Arrowheads indicate representative melanocytes
scattered throughout the ectopic tissue.
Fig. 4. Whole-mount analysis of secondary axes. (A) tBRII RNAinjected
double-axis embryo. Black arrows and white arrowheads
denote primary and secondary axes, respectively. Cement glands
(black arrowheads) are present in both axes, but the secondary axis
lacks recognizable head structures. tBRII injected embryos were
analyzed by in situ hybridization (B,C, E-H) or immunohistochemistry
(D) to determine the tissue types present in the secondary axis. Arrows
and arrowheads (B-H) indicate primary and secondary axes,
respectively. (B) General neural marker, nrp-1. (C) Xbra, notochord
and tailbud marker. The secondary axis lacks expression. (D) 12/101
muscle-specific antibody. (E) HoxB9, spinal cord marker. (F) En2,
midbrain/hindbrain boundary marker. (G) Krox20, marker of
rhombomeres 3 and 5. (H) Otx2, anterior neural marker. Embryos are
oriented with anterior to the left and dorsal up.
Fig. 5. Direct anterior neural induction in animal caps by tBRII. (A)
RNAse protection analysis of animal cap RNA (10 caps/lane) for
neural (NCAM) and mesodermal (Xbra) tissues at stages 25 (tailbud)
and 11 (gastrula) respectively. tBRII induces neural tissue without
mesoderm induction. EF1a is the loading control. WE, whole
embryo control; uninj., control caps from uninjected embryos (B)
RT-PCR analysis of animal cap RNA for region-specific neural
marker expression. tBRII induces the anterior neural marker, Otx2,
but not the hindbrain marker, Krox20. FGFR expression was used as
a loading control. -RT, no reverse transcriptase control.
Fig. 6. tBRII dorsalizes ventral marginal zones. Dorsal and ventral
marginal zones (DMZ and VMZ) were analyzed at stage 12.5 (early)
or stage 25 (late) by RT-PCR for expression of the mesodermal
markers gsc, Xwnt8 and actin. As expected, DMZs show similar
marker expression profiles among the three experimental groups.
tBRII or tBR-loaded VMZs express the dorsal markers gsc and actin,
and the ventral marker, Xwnt8, is suppressed. FGFR is the loading
control. WE, RNA from whole embryos; uninj, DMZs or VMZs
from uninjected embryos; -RT, no reverse transcriptase control.
Fig. 7. tBRII blocks BMP4-mediated mesoderm induction. RT-PCR
analysis of animal cap RNA at stage 16 for expression of the panmesodermal
marker, Xbra, or ventrolateral mesodermal marker,
Xhox3. When compared to control caps (lane 3), incubation of
uninjected animal caps with rhBMP4 induces mesodermal markers
(lane 5). The induction is blocked by tBRII (lane 6) or ÆXAR1 (lane
9). The inhibition is reversed by coexpressing the wild-type BMP
type II (lanes 7 and 11) or activin type II (lanes 8 and 10) receptor.
FGFR expression was used as the loading control. WE, RNA from
whole embryos; uninj, animal caps from uninjected embryos; -RT,
no reverse transcriptase control.
Fig. 8. tBRII does not block activin signaling. (A) Morphological
analysis of animal caps at stage 22. Uninjected animal caps elongate
in response to 5 ng/ml activin (top two panels). This elongation is not
affected by tBRII (next four panels) but is diminished greatly by
ÆXAR1 (bottom four panels). (B) RT-PCR analysis of animal cap
RNA at sibling stage 11 (early) or 25 (late) for expression of the panmesodermal
marker, Xbra, or the dorsolateral mesodermal marker,
actin. Neither marker is significantly expressed in control or tBRII
injected caps (lanes 3-5), but both are induced by treating uninjected
caps with activin (lane 6). Marker expression is not inhibited by tBRII
(lanes 7 and 8), but ÆXAR1 dose-dependently blocks expression
(lanes 9 and 10). FGFR expression was used as a loading control.
WE, RNA from whole embryos; uninj, animal caps from uninjected
embryos; act, activin; -RT, no reverse transcriptase control.
bmpr2 (bone morphogenetic protein receptor, type 2 (serine/threonine kinase)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.