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Fig. 3. BMP-3b and BMP-3 have apparently different dorsalizing activities in Xenopus embryos. (A–C) Xenopus embryos injected with BMP-3b or BMP-3
mRNA. Overexpression of BMP-3b led to ectopic head formation, whereas BMP-3 generated aberrant tail formation. Synthetic mRNAs (1 ng each) encoding
xBMP-3b (A), xBMP-3 (B), or -galactosidase ( -gal; C) were injected into marginal zone of two ventral blastomeres at the four cell stage. (D–G)
Differentiation of ventral marginal zone explants (VMZs) injected with BMP-3b or BMP-3. Typical explants are shown in circles. (D) VMZs injected with
rBMP-3b RNA generated head-like structures, consisting of an eye and cement glands (68%, n 15). (E) VMZs injected with rBMP-3 elongated and formed
melanocytes (93%, n 15). (F) Control VMZs developed cylindrical structures. (G) These VMZs were harvested at stage 41 and scored by RT-PCR.
Injection of BMP-3b and BMP-3 caused expression of dorsal markers (cardiac actin and NCAM), and repression of a ventral marker ( globin). BMP-3b
also elevated anterior tissue markers such as cement gland (xAG1), lens ( B1-crystallin), and retina (rhodopsin). EF1 is an internal loading control. Emb
RT and RT are controls for cDNA synthesis and for genomic DNA contamination, respectively. (H) Analysis of early markers in embryos coinjected
with -gal (100 pg), and either BMP-3b or BMP-3 (1 ng). Light blue staining represents area injected with BMP-3b (left) or BMP-3 (center). Brown staining
represents expression of Otx2 (top panel), and Xwnt-8 (middle panel), and cerberus (bottom panel). All panels are vegetal views of embryos with dorsal aspect
top. Injection of BMP-3b mRNA triggered ectopic Otx2 expression (top left), whereas injection of BMP-3b and BMP-3 reduced endogenous Xwnt8
expression (middle). BMP-3b did not induce cerberus expression, indicating that BMP-3b is involved in head formation independently of, or downstream
from, cerberus. |
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Fig. 9. Repression of xBMP-3b and cerberus leads to impaired gastrulation
movements. Lineage of cells targeted by MOs and expression of
marker genes were monitored at gastrula, neurula and tadpole stages.
(Left) Embryos injected with MOs for both xBMP-3b and cerberus.
(Right) Control embryos injected with globin MO. Light blue staining
represents cells targeted by MOs. Brown staining represents expression
of markers as indicated on right corner. Dorsal side faces top in panels
of gastrula and tadpole, and anterior side faces top in panels of neurula.
Injection of MOs for xBMP-3b and cerberus did not affect dorsal lip
formation and expression of organizer genes at beginning of gastrulation,
but led to delayed blastopore closure and downregulation of Otx2
expression by neurula stage. Consequently, suppressing xBMP-3b and
cerberus functions elevated expression of a ventral–posterior marker,
Xcad1. |
