45
Signaling Components in D-V Patterning
mouse organizer was not known. When I presented the f rst
in situ hybridizations of mouse goosecoid in 1992 at the f rst
vertebrate molecular embryology meeting in Les Diablerets,
Switzerland, great consternation was caused. Our collaborator Stephen Gaunt had found goosecoid expression in
the anterior primitive streak. Mouse embryologists almost
unanimously rose to counter that the organizer was located
in the node that forms posterior to the notochord at a later
stage of development. Fortunately, on the train down from
the mountain, I sat, dejected, next to Azim Surani, who suggested that if the mouse organizer were where we thought it
was, transplantation into the Xenopus blastula cavity might
reveal its inductive activity. We did the experiment with
Martin Blum and Herbert Steinbeisser, and that was the
case; goosecoid has pinpointed the location of the organizer
in many vertebrate embryos since (De Robertis, 2004).
4.3. THE VENTRAL SIGNALING CENTER
The dorsal organizer has been the center of attention, but it
is emerging that the ventral side of the gastrula is equally
important. For each action on the dorsal side, there is a reaction on the ventral side. Ventral genes are turned on by BMP4
signaling, while dorsal genes are transcribed when BMP signaling levels are low (Karaulanov et al., 2004; Reversade and
De Robertis, 2005 ). This transcriptional seesaw explains to a
large degree the self-regulation and resilience of the Xenopus
embryo. The reason the ventral center was ignored for a long
time was that when transplanted, ventral tissue becomes
incorporated into the host site instead of inducing changes
in the neighboring tissues (Spemann, 1938). However, when
BMP2/4/7 are depleted simultaneously so that no epidermis
is formed, transplantation of wild-type ventral tissue can
induce epidermal differentiation (high BMP) at a great distance (Reversade and De Robertis, 2005).
Xenopus BMP4 was found to be expressed in the ventral region and to induce ventralization (Fainsod et al.,
1994). However, the realization that the ventral side serves
to antagonize the effects of the organizer was not formulated until Christof Niehrs discovered Xvent-1 and Xvent2, two homeobox target genes of BMP4 with similarities to
Drosophila bar (Gawantka et al., 1995). It was later found
that these genes mediate the effects of BMP4 (Ladher et al.,
1996; Onichtchouk et al., 1996 ). There is a whole panoply
of genes part of the BMP4 synexpression group (Niehrs and
Pollet, 1999) that are transcriptionally activated by BMP4
signaling, such as Id1–4 (inhibitor of differentiation 1–4)
(Karaulanov et al., 2004) and BAMBI (BMP and Activin
Membrane Bound Inhibitor), a transmembrane pseudoreceptor lacking the cytosolic Serine-Threonine kinase domain
(Onichtchouk et al., 1999).
The function of Xvent1/2 as repressors of the Spemann
organizer has been investigated using antisense morpholino
oligonucleotides (MOs). This method of obtaining loss of function can be very effective and specifc in Xenopus embryos. In
zebrafsh, on occasion, MOs may have toxic effects, and this
has greatly confused the feld, as zebrafsh researchers now
demand genetic mutations while negating the many favorable
properties of MOs in Xenopus and many other organisms, for
which we have advocated elsewhere (Blum et al., 2015). Xvent1
and 2 have redundant functions, but when both are depleted,
embryos are strongly dorsalized with expanded heads and
short trunks (Sander et al., 2007). Notably, the expression of
goosecoid is greatly expanded by loss of Xvent1/2. Depletion
of Xenopus Goosecoid with MO resulted in cyclopic embryos
with small heads and enlarged ventral tissues. Unexpectedly,
triple depletion of Xvent1, Xvent2, and Goosecoid rescued
almost completely normal D-V and anterior-posterior (A-P)
development in a variety of assays (Sander et al., 2007). Thus,
it is as if these three genes are dispensable for embryogenesis
and exist to balance deviations from the norm of their counterparts. Xvent1/2 and Goosecoid in Xenopus mediate a remarkable self-adjusting mechanism to ensure a perfect tadpole is
formed time after time.
The most abundant ventral center transcript in the ventral
center is Sizzled (Szl), a divergent sFRP (secreted frizzledrelated protein) that lost its ability to bind Wnts (Collavin and
Kirschner, 2003; Ding et al., 2017a). Sizzled, like Xolloidrelated protease (Piccolo et al., 1997 ), Twisted gastrulation
(Tsg) (Oelgeschläger et al., 2000), and Crossveinless-2 (CV-2) (
Ambrosio et al., 2008) are ventral center-secreted proteins
that function in the Chordin morphogenetic pathway.
4.4. BMP ANTAGONISTS AND TISSUE
DIFFERENTIATION
The Spemann organizer directs the differentiation of dorsal
tissues. Xenopus is ideal for the analysis of D-V histotypic cell
differentiation because the embryo undergoes a cortical rotation at the one-cell stage that displaces the maternal pigment
towards the ventral side (which is the sperm entry side). If
one pays close attention, the rotation continues at the two- and
four-cell stage. The result is that if one selects symmetrically
dividing embryos, at the four-cell stage, the two blastomeres
containing the less pigmented dorsal crescent will reliably
mark the formation of the dorsal blastopore lip and the embryonic midline (Klein, 1987). This is a very powerful tool that
allows embryologists to direct D-V microinjections, lineage
tracing, and transplantations from the earliest stages of development. Surprisingly, this useful tool was disputed by lineage
tracing random embryos, which resulted in the publication of
a paper (Danilchik and Black, 1988), but, fortunately, it did
not stop further lineage-tracing research in Xenopus. It is
good that today some journals are starting to publish papers
confrming, not only negating, previous observations.
At the 16-cell stage, the Xenopus embryo has a
predictable cell lineage (Moody, 1987). At this stage,
four segments can be distinguished (S1–S4) on each side,
which can be marked individually by four microinjections
of fuorescent lineage tracers (Moriyama and De Robertis,
2018). In the beautiful embryo shown in Figure 4.2, the
descendants of the organizer can be followed in red and
progressively more ventral tissues in green, blue, and f nally
orange. This tour-de-force by Yuki Moriyama reveals that
Signaling Components in D-V Patterning
mouse organizer was not known. When I presented the f rst
in situ hybridizations of mouse goosecoid in 1992 at the f rst
vertebrate molecular embryology meeting in Les Diablerets,
Switzerland, great consternation was caused. Our collaborator Stephen Gaunt had found goosecoid expression in
the anterior primitive streak. Mouse embryologists almost
unanimously rose to counter that the organizer was located
in the node that forms posterior to the notochord at a later
stage of development. Fortunately, on the train down from
the mountain, I sat, dejected, next to Azim Surani, who suggested that if the mouse organizer were where we thought it
was, transplantation into the Xenopus blastula cavity might
reveal its inductive activity. We did the experiment with
Martin Blum and Herbert Steinbeisser, and that was the
case; goosecoid has pinpointed the location of the organizer
in many vertebrate embryos since (De Robertis, 2004).
4.3. THE VENTRAL SIGNALING CENTER
The dorsal organizer has been the center of attention, but it
is emerging that the ventral side of the gastrula is equally
important. For each action on the dorsal side, there is a reaction on the ventral side. Ventral genes are turned on by BMP4
signaling, while dorsal genes are transcribed when BMP signaling levels are low (Karaulanov et al., 2004; Reversade and
De Robertis, 2005 ). This transcriptional seesaw explains to a
large degree the self-regulation and resilience of the Xenopus
embryo. The reason the ventral center was ignored for a long
time was that when transplanted, ventral tissue becomes
incorporated into the host site instead of inducing changes
in the neighboring tissues (Spemann, 1938). However, when
BMP2/4/7 are depleted simultaneously so that no epidermis
is formed, transplantation of wild-type ventral tissue can
induce epidermal differentiation (high BMP) at a great distance (Reversade and De Robertis, 2005).
Xenopus BMP4 was found to be expressed in the ventral region and to induce ventralization (Fainsod et al.,
1994). However, the realization that the ventral side serves
to antagonize the effects of the organizer was not formulated until Christof Niehrs discovered Xvent-1 and Xvent2, two homeobox target genes of BMP4 with similarities to
Drosophila bar (Gawantka et al., 1995). It was later found
that these genes mediate the effects of BMP4 (Ladher et al.,
1996; Onichtchouk et al., 1996 ). There is a whole panoply
of genes part of the BMP4 synexpression group (Niehrs and
Pollet, 1999) that are transcriptionally activated by BMP4
signaling, such as Id1–4 (inhibitor of differentiation 1–4)
(Karaulanov et al., 2004) and BAMBI (BMP and Activin
Membrane Bound Inhibitor), a transmembrane pseudoreceptor lacking the cytosolic Serine-Threonine kinase domain
(Onichtchouk et al., 1999).
The function of Xvent1/2 as repressors of the Spemann
organizer has been investigated using antisense morpholino
oligonucleotides (MOs). This method of obtaining loss of function can be very effective and specifc in Xenopus embryos. In
zebrafsh, on occasion, MOs may have toxic effects, and this
has greatly confused the feld, as zebrafsh researchers now
demand genetic mutations while negating the many favorable
properties of MOs in Xenopus and many other organisms, for
which we have advocated elsewhere (Blum et al., 2015). Xvent1
and 2 have redundant functions, but when both are depleted,
embryos are strongly dorsalized with expanded heads and
short trunks (Sander et al., 2007). Notably, the expression of
goosecoid is greatly expanded by loss of Xvent1/2. Depletion
of Xenopus Goosecoid with MO resulted in cyclopic embryos
with small heads and enlarged ventral tissues. Unexpectedly,
triple depletion of Xvent1, Xvent2, and Goosecoid rescued
almost completely normal D-V and anterior-posterior (A-P)
development in a variety of assays (Sander et al., 2007). Thus,
it is as if these three genes are dispensable for embryogenesis
and exist to balance deviations from the norm of their counterparts. Xvent1/2 and Goosecoid in Xenopus mediate a remarkable self-adjusting mechanism to ensure a perfect tadpole is
formed time after time.
The most abundant ventral center transcript in the ventral
center is Sizzled (Szl), a divergent sFRP (secreted frizzledrelated protein) that lost its ability to bind Wnts (Collavin and
Kirschner, 2003; Ding et al., 2017a). Sizzled, like Xolloidrelated protease (Piccolo et al., 1997 ), Twisted gastrulation
(Tsg) (Oelgeschläger et al., 2000), and Crossveinless-2 (CV-2) (
Ambrosio et al., 2008) are ventral center-secreted proteins
that function in the Chordin morphogenetic pathway.
4.4. BMP ANTAGONISTS AND TISSUE
DIFFERENTIATION
The Spemann organizer directs the differentiation of dorsal
tissues. Xenopus is ideal for the analysis of D-V histotypic cell
differentiation because the embryo undergoes a cortical rotation at the one-cell stage that displaces the maternal pigment
towards the ventral side (which is the sperm entry side). If
one pays close attention, the rotation continues at the two- and
four-cell stage. The result is that if one selects symmetrically
dividing embryos, at the four-cell stage, the two blastomeres
containing the less pigmented dorsal crescent will reliably
mark the formation of the dorsal blastopore lip and the embryonic midline (Klein, 1987). This is a very powerful tool that
allows embryologists to direct D-V microinjections, lineage
tracing, and transplantations from the earliest stages of development. Surprisingly, this useful tool was disputed by lineage
tracing random embryos, which resulted in the publication of
a paper (Danilchik and Black, 1988), but, fortunately, it did
not stop further lineage-tracing research in Xenopus. It is
good that today some journals are starting to publish papers
confrming, not only negating, previous observations.
At the 16-cell stage, the Xenopus embryo has a
predictable cell lineage (Moody, 1987). At this stage,
four segments can be distinguished (S1–S4) on each side,
which can be marked individually by four microinjections
of fuorescent lineage tracers (Moriyama and De Robertis,
2018). In the beautiful embryo shown in Figure 4.2, the
descendants of the organizer can be followed in red and
progressively more ventral tissues in green, blue, and f nally
orange. This tour-de-force by Yuki Moriyama reveals that