47
Signaling Components in D-V Patterning
depleted, indicating an essential role of Chordin in the dorsalization of mesoderm (Oelgeschläger et al., 2003).
4.5. NEURAL INDUCTION BY THE ORGANIZER
Xenopus blastula ectodermal explants, also called animal
caps, provide an ideal system to study the induction of the
CNS, which has fascinated biologists since Spemann (De
Robertis and Kuroda, 2004). Animal cap explants develop
into epidermis and contain high levels of BMP signaling
( Wilson and Hemmati-Brivanlou, 1995). Treatment of animal cap cells with Noggin protein induced anterior brain tissue (Lamb et al., 1993). Similarly, microinjection of mRNAs
encoding Chordin, Noggin, or Follistatin also induced anterior neural tissue (Sasai et al., 1995; Kuroda et al., 2004).
Neural tissue is the default state of the animal cap, and
when cells are dissociated in low calcium, they become
neural due to sustained activation of the Ras/MAPK pathway (Kuroda et al., 2005 ). MAPK phosphorylation primes
the BMP transcriptional regulators Smad1/5/8 for phosphorylation by GSK3 and subsequent degradation, resulting in reduced BMP signaling (Fuentealba et al., 2007).
Neural induction requires the inhibition of both the BMP
(Smad1) and TGF-β (Smad2) pathways (Chang and Harland,
2007). This can be achieved through polyubiquitinylation
triggered by FGF/MAPK/GSK3 phosphorylations of the
shared Smad4 subunit (which is not a substrate for SerineThreonine kinase receptors) (Demagny et al., 2014). Modern
studies of the Spemann organizer have greatly helped formulate current models of the regulation of CNS induction.
4.6. THE CHORDIN/TOLLOID/TWISTED
GASTRULATION/CROSSVEINLESS-2/
BMP ANCESTRALLY CONSERVED
D-V PATTERNING SYSTEM
Chordin is at the center of D-V patterning and is a key component of a biochemical pathway of interacting extracellular
proteins in Xenopus ( Figure 4.1B). Chordin facilitates the
diffusion (or fux) of dorsal BMPs towards the ventral side of
the embryo, where they are released by a specif c chordinase
called Tolloid (Xolloid-related in Xenopus ) ( Piccolo et al.,
1997 ). At the highest levels of BMP signaling, Sizzled is
secreted at levels comparable to those of Chordin
( Lee et al., 2006). Sizzled functions as a competitive
inhibitor of Tolloid, as it is bound by the active site of the
protease but cannot be cut (Lee et al., 2006). However,
Tolloid enzyme activity is also non-competitively inhibited
by the binding of BMP to the so-called CUB domains of this
metalloproteinase (Lee et al., 2009). Chordin and Sizzled
are the highest enriched transcripts both at the dorsal and
ventral poles of the embryo (Ding et al., 2017a).
The Chordin/Tolloid system is self-regulating and
adjustable to size-dependent scaling (Ben-Zvi et al., 2008;
Inomata et al., 2013). Diffusion of Chordin/BMP driven by
copious degradation by Tolloid takes place in the narrow
region of extracellular matrix that separates ectoderm from
mesoderm, called Brachet’s cleft in Xenopus ( Plouhinec
et al., 2013). Tsg secreted ventrally forms a ternary complex
with BMP and Chordin, facilitating the transfer of
BMP to its cell-surface receptors after Tolloid cleavage
(Oelgeschläger et al., 2000; Zinski et al., 2018). CV2 does
not diffuse but serves as a binding site to attract Chd/Tsg/
BMP to the ventral side for cleavage by Tolloid and release
of BMP/Tsg (Ambrosio et al., 2008) (Figure 4.1B). This
D-V patterning pathway is strongly supported by loss-offunction mutations in zebrafsh (Little and Mullins, 2006;
Zinski et al., 2018).
Chordin is the homologue of Drosophila Short
gastrulation (Sog) (Holley et al., 1995). Tsg, Tolloid, and
CV2, but not Sizzled, have Drosophila homologues as well.
In zebrafsh, Sizzled is called Ogon/Mercedes (Little and
Mullins, 2006). As reviewed elsewhere, the remarkable Chd/Tolloid/Tsg/CV2/BMP biochemical pathway is
ancestral to bilateral animals and is even conserved in the
sea anemone Nematostella (Bier and De Robertis, 2015;
De Robertis et al., 2017).
4.7. SELF-REGULATION BY SPEMANN
ORGANIZER RELOCALIZATION
Spemann had been able to obtain twins by hair-loop constrictions of the fertilized egg (Spemann, 1938). Many years
later, we realized that identical twins could be obtained in
Xenopus by cutting blastula embryos sagittally with a scalpel
blade, but this occurred only at very low frequencies. More
recently, the frequency of twinning was greatly improved
by bisecting blastula embryos with an eyelash knife and
improving culture conditions. With frequencies of twinning
of 50% or more, it became possible to follow the molecular
changes that take place when the entire missing half of the
embryo is regenerated (Moriyama and De Robertis, 2018).
Figure 4.3 shows the results of sagittal or dorsal/ventral
bisections. We unexpectedly found that, in twins that healed
properly, the maternal egg pigmentation was routinely
asymmetric on the left or right sides (Figure 4.3B’’ and C ’’),
prompting a series of in-depth lineage tracing investigations. The half embryo heals the large wound left by bisection within 60 minutes, bringing the most dorsal segment
1 fated to become the Spemann organizer in direct contact
with the ventral-most cells of segment 4. From this opposition, the formation of the dorsal organizer, which is not
yet established at mid blastula, is displaced by 90° (Figure
4.3B’ and C ’). This explains the pigment asymmetry, for the
most pigmented epidermis arises from the ventral-most segment 4 after the mesoderm involutes forming the left and
right side. Studies using Chordin and phospho-Smad1/5/8
confrmed that a new D-V gradient is formed by repositioning the D-V axis, explaining self-organization of twins after
bisection (Moriyama and De Robertis, 2018). When bisection is performed perpendicularly to the sagittal plane, the
dorsal half can scale the size of the gradient into an approximately normal embryo (Figure 4.3D–D’’), but the ventral
half forms only ventral mesoderm, as it lacks a Spemann
Signaling Components in D-V Patterning
depleted, indicating an essential role of Chordin in the dorsalization of mesoderm (Oelgeschläger et al., 2003).
4.5. NEURAL INDUCTION BY THE ORGANIZER
Xenopus blastula ectodermal explants, also called animal
caps, provide an ideal system to study the induction of the
CNS, which has fascinated biologists since Spemann (De
Robertis and Kuroda, 2004). Animal cap explants develop
into epidermis and contain high levels of BMP signaling
( Wilson and Hemmati-Brivanlou, 1995). Treatment of animal cap cells with Noggin protein induced anterior brain tissue (Lamb et al., 1993). Similarly, microinjection of mRNAs
encoding Chordin, Noggin, or Follistatin also induced anterior neural tissue (Sasai et al., 1995; Kuroda et al., 2004).
Neural tissue is the default state of the animal cap, and
when cells are dissociated in low calcium, they become
neural due to sustained activation of the Ras/MAPK pathway (Kuroda et al., 2005 ). MAPK phosphorylation primes
the BMP transcriptional regulators Smad1/5/8 for phosphorylation by GSK3 and subsequent degradation, resulting in reduced BMP signaling (Fuentealba et al., 2007).
Neural induction requires the inhibition of both the BMP
(Smad1) and TGF-β (Smad2) pathways (Chang and Harland,
2007). This can be achieved through polyubiquitinylation
triggered by FGF/MAPK/GSK3 phosphorylations of the
shared Smad4 subunit (which is not a substrate for SerineThreonine kinase receptors) (Demagny et al., 2014). Modern
studies of the Spemann organizer have greatly helped formulate current models of the regulation of CNS induction.
4.6. THE CHORDIN/TOLLOID/TWISTED
GASTRULATION/CROSSVEINLESS-2/
BMP ANCESTRALLY CONSERVED
D-V PATTERNING SYSTEM
Chordin is at the center of D-V patterning and is a key component of a biochemical pathway of interacting extracellular
proteins in Xenopus ( Figure 4.1B). Chordin facilitates the
diffusion (or fux) of dorsal BMPs towards the ventral side of
the embryo, where they are released by a specif c chordinase
called Tolloid (Xolloid-related in Xenopus ) ( Piccolo et al.,
1997 ). At the highest levels of BMP signaling, Sizzled is
secreted at levels comparable to those of Chordin
( Lee et al., 2006). Sizzled functions as a competitive
inhibitor of Tolloid, as it is bound by the active site of the
protease but cannot be cut (Lee et al., 2006). However,
Tolloid enzyme activity is also non-competitively inhibited
by the binding of BMP to the so-called CUB domains of this
metalloproteinase (Lee et al., 2009). Chordin and Sizzled
are the highest enriched transcripts both at the dorsal and
ventral poles of the embryo (Ding et al., 2017a).
The Chordin/Tolloid system is self-regulating and
adjustable to size-dependent scaling (Ben-Zvi et al., 2008;
Inomata et al., 2013). Diffusion of Chordin/BMP driven by
copious degradation by Tolloid takes place in the narrow
region of extracellular matrix that separates ectoderm from
mesoderm, called Brachet’s cleft in Xenopus ( Plouhinec
et al., 2013). Tsg secreted ventrally forms a ternary complex
with BMP and Chordin, facilitating the transfer of
BMP to its cell-surface receptors after Tolloid cleavage
(Oelgeschläger et al., 2000; Zinski et al., 2018). CV2 does
not diffuse but serves as a binding site to attract Chd/Tsg/
BMP to the ventral side for cleavage by Tolloid and release
of BMP/Tsg (Ambrosio et al., 2008) (Figure 4.1B). This
D-V patterning pathway is strongly supported by loss-offunction mutations in zebrafsh (Little and Mullins, 2006;
Zinski et al., 2018).
Chordin is the homologue of Drosophila Short
gastrulation (Sog) (Holley et al., 1995). Tsg, Tolloid, and
CV2, but not Sizzled, have Drosophila homologues as well.
In zebrafsh, Sizzled is called Ogon/Mercedes (Little and
Mullins, 2006). As reviewed elsewhere, the remarkable Chd/Tolloid/Tsg/CV2/BMP biochemical pathway is
ancestral to bilateral animals and is even conserved in the
sea anemone Nematostella (Bier and De Robertis, 2015;
De Robertis et al., 2017).
4.7. SELF-REGULATION BY SPEMANN
ORGANIZER RELOCALIZATION
Spemann had been able to obtain twins by hair-loop constrictions of the fertilized egg (Spemann, 1938). Many years
later, we realized that identical twins could be obtained in
Xenopus by cutting blastula embryos sagittally with a scalpel
blade, but this occurred only at very low frequencies. More
recently, the frequency of twinning was greatly improved
by bisecting blastula embryos with an eyelash knife and
improving culture conditions. With frequencies of twinning
of 50% or more, it became possible to follow the molecular
changes that take place when the entire missing half of the
embryo is regenerated (Moriyama and De Robertis, 2018).
Figure 4.3 shows the results of sagittal or dorsal/ventral
bisections. We unexpectedly found that, in twins that healed
properly, the maternal egg pigmentation was routinely
asymmetric on the left or right sides (Figure 4.3B’’ and C ’’),
prompting a series of in-depth lineage tracing investigations. The half embryo heals the large wound left by bisection within 60 minutes, bringing the most dorsal segment
1 fated to become the Spemann organizer in direct contact
with the ventral-most cells of segment 4. From this opposition, the formation of the dorsal organizer, which is not
yet established at mid blastula, is displaced by 90° (Figure
4.3B’ and C ’). This explains the pigment asymmetry, for the
most pigmented epidermis arises from the ventral-most segment 4 after the mesoderm involutes forming the left and
right side. Studies using Chordin and phospho-Smad1/5/8
confrmed that a new D-V gradient is formed by repositioning the D-V axis, explaining self-organization of twins after
bisection (Moriyama and De Robertis, 2018). When bisection is performed perpendicularly to the sagittal plane, the
dorsal half can scale the size of the gradient into an approximately normal embryo (Figure 4.3D–D’’), but the ventral
half forms only ventral mesoderm, as it lacks a Spemann