102
M. Hild et al.
amounts of zebrafish or Xenopus bmp2 or bmp4 mRNA were injected,
the dorsalization could be significantly normalized (from C4 to Cl), and
even converted to a ventralization upon injection of high amounts of
bmp214 mRNA (C4 to V3).
To determine at what time point during development exogenous
Bmp2/4 can override the requirement for smad5, human bmp4 was
expressed in sbn smad5 mutant embryos under the control of the cyto skeletal actin promoter. In contrast to the corresponding smad5 transgene (see above), expression of the human bmp4 gene under this promoter led to a striking rescue and even ventralization of sbn mutant
embryos, suggesting that the smad5-independent response of the zebrafish embryo to exogenous Bmp2/4 occurs during gastrula stages.
In the bmp214 RNA injection experiments described above, the actual in vivo concentrations of Bmp2/4 protein are unknown. To investigate whether sbn cells can respond to Bmp2/4 under physiological
conditions, cell transplantation experiments were carried out. Labeled
sbn smad5 mutant cells were transplanted into wild-type embryos. In all
cases, donor cells gave rise to blood and ventral tail fin, ventrally
derived tissues that are completely absent in sbn mutant embryos. The
frequencies of ventral tissue contribution were similar, independently of
whether donor cells were wild-type or sbn mutant, and whether they
were transplanted at late blastula or early gastrula stages. Together with
the Bmp2/4 injection experiments, these data indicate that in contrast to
the early mediation of Bmp2/4 signaling, the final specification of
ventral cell fates can occur in the absence of functional Smad5.
Altogether, the behavior of the sbn smad5 and swr bmp2b mutations
define three distinct phases of dorsoventral patterning. In the first phase,
an initial coarse dorsoventral pattern is set up at a time when the future
dorsal mesoderm, the equivalent of the amphibian Spemann organizer,
is induced in a small dorsal marginal domain characterized by the
expression of chordino and opposed by the expression of bmp2b in the
rest of the embryo. This pattern, which is most likely set up under
maternal control, is independent of Bmp2b and Smad5, although in
Xenopus, Bmp2/4 and Smad1l5 have been discussed as candidate maternal components involved in the induction of ventral mesoderm (Graff
et al. 1996).
In the second phase, which is under zygotic control and dependent on
Bmp2b and Smad5, the initial dorsoventral pattern is refined, leading to
M. Hild et al.
amounts of zebrafish or Xenopus bmp2 or bmp4 mRNA were injected,
the dorsalization could be significantly normalized (from C4 to Cl), and
even converted to a ventralization upon injection of high amounts of
bmp214 mRNA (C4 to V3).
To determine at what time point during development exogenous
Bmp2/4 can override the requirement for smad5, human bmp4 was
expressed in sbn smad5 mutant embryos under the control of the cyto skeletal actin promoter. In contrast to the corresponding smad5 transgene (see above), expression of the human bmp4 gene under this promoter led to a striking rescue and even ventralization of sbn mutant
embryos, suggesting that the smad5-independent response of the zebrafish embryo to exogenous Bmp2/4 occurs during gastrula stages.
In the bmp214 RNA injection experiments described above, the actual in vivo concentrations of Bmp2/4 protein are unknown. To investigate whether sbn cells can respond to Bmp2/4 under physiological
conditions, cell transplantation experiments were carried out. Labeled
sbn smad5 mutant cells were transplanted into wild-type embryos. In all
cases, donor cells gave rise to blood and ventral tail fin, ventrally
derived tissues that are completely absent in sbn mutant embryos. The
frequencies of ventral tissue contribution were similar, independently of
whether donor cells were wild-type or sbn mutant, and whether they
were transplanted at late blastula or early gastrula stages. Together with
the Bmp2/4 injection experiments, these data indicate that in contrast to
the early mediation of Bmp2/4 signaling, the final specification of
ventral cell fates can occur in the absence of functional Smad5.
Altogether, the behavior of the sbn smad5 and swr bmp2b mutations
define three distinct phases of dorsoventral patterning. In the first phase,
an initial coarse dorsoventral pattern is set up at a time when the future
dorsal mesoderm, the equivalent of the amphibian Spemann organizer,
is induced in a small dorsal marginal domain characterized by the
expression of chordino and opposed by the expression of bmp2b in the
rest of the embryo. This pattern, which is most likely set up under
maternal control, is independent of Bmp2b and Smad5, although in
Xenopus, Bmp2/4 and Smad1l5 have been discussed as candidate maternal components involved in the induction of ventral mesoderm (Graff
et al. 1996).
In the second phase, which is under zygotic control and dependent on
Bmp2b and Smad5, the initial dorsoventral pattern is refined, leading to