56
Xenopus
different time points. Stimulation of FGF signaling during
mid-gastrulation could still lead to induction of posterior
markers ( Pownall et al., 2003). Furthermore, transgenic
approaches have been used to block FGF signaling during
gastrulation and neurulation, with the results supporting a
function of FGF in posterior development (Pownall et al.,
1998). A spliced isoform of fgf8, fgf8a, was further found
to have minimal mesodermal inducing ability but was fully
active in inducing posterior neural markers. Knockdown of
this isoform did not affect mesodermal marker expression
at gastrula stages but reduced expression of posterior neural
genes in neurula stage embryos (Christen and Slack, 1997;
Fletcher et al., 2006). Taken together, the data demonstrate
that FGF signaling can act at two temporal phases to induce
mesoderm and posteriorize neural tissues, respectively.
Another issue concerning FGF as a caudalizing factor is
that while XFD blocked posterior hox neural gene induction by FGF in explants neuralized with BMP inhibitors, it
had variable effects in preventing the induction of these hox
genes in animal caps conjugated with the wild-type organizer (Curran and Grainger, 2000; Holowacz and Sokol,
1999). This implied that redundant endogenous signals could
operate in the absence of FGF to posteriorize neural tissues.
As we now know, other signals indeed work in parallel and
cooperate with FGF to caudalize neural tissues.
A perhaps more serious issue is centered around whether
FGF can directly induce posterior neural markers. Based
on the model proposed by Mangold, there should be separate inducers for anterior and posterior neural tissues
(Doniach, 1993; Mangold, 1933; Slack and Tannahill,
1992). However, if the activation-transformation model
proposed by Nieuwkoop is in operation, the transforming
agent cannot activate the neural program alone and should
only change the characteristics of the neural tissues already
induced by an activator present along the entire AP axis
( Nieuwkoop, 1952a , 1952b , 1952c ). Several groups reported
that FGF induced both pan-neural markers and posterior
neural genes in animal cap explants in the absence of mesoderm (Kengaku and Okamoto, 1995; Lamb and Harland,
1995). However, these studies seemed to have used explants
that were either dissociated or kept partially open by a low
calcium medium prior to the treatment with FGF. It is well
documented that explants raised under these conditions
experience decreased BMP signaling (Grunz and Tacke,
1989; Sato and Sargent, 1989). Therefore, these explants
might have already been primed toward a neural state,
which could then be changed into the posterior neural characters by FGF. Loss-of-function experiments using XFD/
DN-Fgfr1 or DN-Ras revealed that it did not block panneural markers, but another DN FGF receptor, DN-Fgfr4a,
was shown to reduce neural induction by organizer or cell
dissociation (Holowacz and Sokol, 1999; Hongo et al.,
1999; Ribisi et al., 2000). The data imply that distinct FGF
ligands and receptors may be involved in neural induction
or maintenance in particular embryonic regions, whereas
other FGF ligand/receptor pairs may play more crucial
roles in neural caudalization.
The idea that the FGF pathway does not simply act as a
graded posteriorizing signal is also suggested by the observation that interference of the pathway not only led to posterior truncation but also induced specifc defects in sensory
organs and/or head organization. The malformation was
consistent with the expression patterns of FGF ligands and
receptors, with many of them found in specifc domains in
the head regions from neurula stages onward (Lea et al.,
2009). Hence, although FGF signaling is essential for posterior development, the specifc ligand and receptor complexes
expressed in different spatial positions and different developmental times infuence the outcome of experiments that
address AP neural specif cation.
5.3.3. WNT/β-CATENIN SIGNALING PATHWAY
The intensive exploration of growth factor signaling in
early vertebrate development before the turn of the 21st
century revealed that Wnt/β-Catenin signaling pathway was
crucial for dorsal cell fate determination and could induce
a secondary axis when activated in the ventral tissues of
cleavage-stage embryos (Christian et al., 1991; McMahon
and Moon, 1989; Smith and Harland, 1991; Sokol et al.,
1991). Interestingly, it was reported that the opposite effects
on embryonic development could be obtained depending on
the timing of Wnt signal stimulation. Ectopic expression of
Wnt/β-Catenin pathway components from mRNAs, which
could be translated into proteins soon after injection, led to
a dorsalized phenotype, whereas expression of the same signaling components from plasmids, which were transcribed
and translated after the mid-blastula transition (MBT),
resulted in head truncation (Christian et al., 1991; Christian
and Moon, 1993; Darken and Wilson, 2001; Fredieu et
al., 1997 ). These data pointed to the distinct functions of
Wnt signaling during embryogenesis, with an early phase
involved in dorsal-ventral patterning and a late phase in
head suppression. A role of Wnt signaling in AP patterning was further supported both by expression patterns of
Wnt pathway components and by functional manipulation
of Wnt signaling levels. Ectopic expression, knockdown, or
dominantly interfering approaches in post-MBT embryos or
in neuralized animal caps and neural plate explants showed
that changing the levels or the activities of Wnt ligands, signal transducers (e.g. Dishevelled), or nuclear transcription
factors (e.g. β-catenin) all led to alterations in AP neural
marker expression or defects in head or trunk structures
(Darken and Wilson, 2001; Domingos et al., 2001; Fredieu
et al., 1997; Itoh and Sokol, 1997; McGrew et al., 1997;
McGrew et al., 1995; Wheeler et al., 2000). These results
suggest that, similarly to RA and FGF signals, the Wnt/βCatenin pathway can act as a transforming factor to specify
posterior neural development.
5.3.3.1. Wnt Pathway in Head Formation
Though AP patterning of the neural tissues is often at the
center of the investigation, key insights about Wnt/β-Catenin
Xenopus
different time points. Stimulation of FGF signaling during
mid-gastrulation could still lead to induction of posterior
markers ( Pownall et al., 2003). Furthermore, transgenic
approaches have been used to block FGF signaling during
gastrulation and neurulation, with the results supporting a
function of FGF in posterior development (Pownall et al.,
1998). A spliced isoform of fgf8, fgf8a, was further found
to have minimal mesodermal inducing ability but was fully
active in inducing posterior neural markers. Knockdown of
this isoform did not affect mesodermal marker expression
at gastrula stages but reduced expression of posterior neural
genes in neurula stage embryos (Christen and Slack, 1997;
Fletcher et al., 2006). Taken together, the data demonstrate
that FGF signaling can act at two temporal phases to induce
mesoderm and posteriorize neural tissues, respectively.
Another issue concerning FGF as a caudalizing factor is
that while XFD blocked posterior hox neural gene induction by FGF in explants neuralized with BMP inhibitors, it
had variable effects in preventing the induction of these hox
genes in animal caps conjugated with the wild-type organizer (Curran and Grainger, 2000; Holowacz and Sokol,
1999). This implied that redundant endogenous signals could
operate in the absence of FGF to posteriorize neural tissues.
As we now know, other signals indeed work in parallel and
cooperate with FGF to caudalize neural tissues.
A perhaps more serious issue is centered around whether
FGF can directly induce posterior neural markers. Based
on the model proposed by Mangold, there should be separate inducers for anterior and posterior neural tissues
(Doniach, 1993; Mangold, 1933; Slack and Tannahill,
1992). However, if the activation-transformation model
proposed by Nieuwkoop is in operation, the transforming
agent cannot activate the neural program alone and should
only change the characteristics of the neural tissues already
induced by an activator present along the entire AP axis
( Nieuwkoop, 1952a , 1952b , 1952c ). Several groups reported
that FGF induced both pan-neural markers and posterior
neural genes in animal cap explants in the absence of mesoderm (Kengaku and Okamoto, 1995; Lamb and Harland,
1995). However, these studies seemed to have used explants
that were either dissociated or kept partially open by a low
calcium medium prior to the treatment with FGF. It is well
documented that explants raised under these conditions
experience decreased BMP signaling (Grunz and Tacke,
1989; Sato and Sargent, 1989). Therefore, these explants
might have already been primed toward a neural state,
which could then be changed into the posterior neural characters by FGF. Loss-of-function experiments using XFD/
DN-Fgfr1 or DN-Ras revealed that it did not block panneural markers, but another DN FGF receptor, DN-Fgfr4a,
was shown to reduce neural induction by organizer or cell
dissociation (Holowacz and Sokol, 1999; Hongo et al.,
1999; Ribisi et al., 2000). The data imply that distinct FGF
ligands and receptors may be involved in neural induction
or maintenance in particular embryonic regions, whereas
other FGF ligand/receptor pairs may play more crucial
roles in neural caudalization.
The idea that the FGF pathway does not simply act as a
graded posteriorizing signal is also suggested by the observation that interference of the pathway not only led to posterior truncation but also induced specifc defects in sensory
organs and/or head organization. The malformation was
consistent with the expression patterns of FGF ligands and
receptors, with many of them found in specifc domains in
the head regions from neurula stages onward (Lea et al.,
2009). Hence, although FGF signaling is essential for posterior development, the specifc ligand and receptor complexes
expressed in different spatial positions and different developmental times infuence the outcome of experiments that
address AP neural specif cation.
5.3.3. WNT/β-CATENIN SIGNALING PATHWAY
The intensive exploration of growth factor signaling in
early vertebrate development before the turn of the 21st
century revealed that Wnt/β-Catenin signaling pathway was
crucial for dorsal cell fate determination and could induce
a secondary axis when activated in the ventral tissues of
cleavage-stage embryos (Christian et al., 1991; McMahon
and Moon, 1989; Smith and Harland, 1991; Sokol et al.,
1991). Interestingly, it was reported that the opposite effects
on embryonic development could be obtained depending on
the timing of Wnt signal stimulation. Ectopic expression of
Wnt/β-Catenin pathway components from mRNAs, which
could be translated into proteins soon after injection, led to
a dorsalized phenotype, whereas expression of the same signaling components from plasmids, which were transcribed
and translated after the mid-blastula transition (MBT),
resulted in head truncation (Christian et al., 1991; Christian
and Moon, 1993; Darken and Wilson, 2001; Fredieu et
al., 1997 ). These data pointed to the distinct functions of
Wnt signaling during embryogenesis, with an early phase
involved in dorsal-ventral patterning and a late phase in
head suppression. A role of Wnt signaling in AP patterning was further supported both by expression patterns of
Wnt pathway components and by functional manipulation
of Wnt signaling levels. Ectopic expression, knockdown, or
dominantly interfering approaches in post-MBT embryos or
in neuralized animal caps and neural plate explants showed
that changing the levels or the activities of Wnt ligands, signal transducers (e.g. Dishevelled), or nuclear transcription
factors (e.g. β-catenin) all led to alterations in AP neural
marker expression or defects in head or trunk structures
(Darken and Wilson, 2001; Domingos et al., 2001; Fredieu
et al., 1997; Itoh and Sokol, 1997; McGrew et al., 1997;
McGrew et al., 1995; Wheeler et al., 2000). These results
suggest that, similarly to RA and FGF signals, the Wnt/βCatenin pathway can act as a transforming factor to specify
posterior neural development.
5.3.3.1. Wnt Pathway in Head Formation
Though AP patterning of the neural tissues is often at the
center of the investigation, key insights about Wnt/β-Catenin