130
Xenopus
across bilaterians. They are also instrumental in specifying a subset of neural crest cells. In Xenopus, morpholinomediated depletion of Twist1 leads to defects in neural crest
specifcation and migration and ultimately results in loss of
neural crest-derived elements of the head skeleton ( Lander
et al. 2011 ). Forced expression of Twist1 in Xenopus also
partially inhibits some aspects of neural crest development,
in part by pushing them toward an ecto-mesenchymal fate at
the expense of non-ectomesenchyme ( Lander et al. 2011 ).
Twist1, along with several other factors (Snai1, Sip1, Hif1a),
regulates EMTs in diverse cell types, including neural crest
in Xenopus ( Linker et al. 2000 ; Lander et al. 2011 ; Barriga
et al. 2013 ). Twist1 mediates this EMT program in part by
physically interacting with and modulating the activity of
Snai1/Snai2 proteins, which is regulated by Gsk3ß-mediated
phosphorylation ( Lander et al. 2013 ).
Snail family transcription factors are also important for
regulating cell migration across metazoans (Nieto 2002). In
most vertebrates, including Xenopus, both Snai1 and Snai2
factors are essential for regulating neural crest EMT (Carl,
Dufton et al. 1999; LaBonne and Bronner-Fraser 2000;
Aybar et al. 2003; Heeg-Truesdell and LaBonne 2004),
although there is evidence that some of their functions are
partially redundant and have been swapped during evolution (Locascio et al. 2002). The mechanisms by which Snai1
and Snai2 control neural crest EMT have been a topic of
intense study. This typically occurs by direct transcriptional
repression of genes that promote epithelial integrity (e.g.
cdh1) (Cano et al. 2000; Langer et al. 2008), which is often
enhanced by physical interaction with other EMT factors,
including the Polycomb repressor complex (Tien et al. 2015),
lmo4 (Ochoa et al. 2012; Ferronha et al. 2013), and Ajuba
LIM-domain proteins (Langer et al. 2008). Moreover, the
ability of Snai1 and Snai2 to exert their effects on neural
crest formation and migration are highly dependent upon
their stability, which is controlled by the F-box protein Ppa
(Vernon and LaBonne 2006; Lander et al. 2011) and can be
stabilized by Elp3 (Yang et al. 2016).
The forkhead box/winged helix transcription factor Foxd3
also plays critical roles in both controlling the neural crest
stem cell state and in promoting EMT. Work in Xenopus has
shown that knockdown of Foxd3 activity reduces expression of neural crest regulatory factors and causes defects in
neural crest migration (Sasai et al. 2001). A similar result
is obtained when foxd3 is ectopically expressed, suggesting
that precise levels of this transcription factor are required
for normal neural crest development (Pohl and Knöchel
2001). Although homologs of forkhead/fox genes are found
in invertebrates, the vertebrate foxd3 paralog is uniquely
suited for specifcation and EMT in the neural crest by having evolved a unique N-terminal sequence (Ono et al. 2014).
Because neural crest cells contribute derivatives throughout the body plan, they must migrate extensively to reach
those sites (Theveneau and Mayor 2012b; Gouignard et al.
2018 ). Xenopus is the only tetrapod model in which this complex process can be studied both in vitro and in vivo , enabling
fne-grained quantitative analysis of the migratory properties
of neural crest cells (Borchers et al. 2000; Alfandari et al.
2010; Toro-Tapia et al. 2017; Barriga et al. 2019). The cranial neural crest in amphibians initiates migration in three
discrete streams along the anterior-posterior axis, termed
mandibular, hyoid, and branchial (Sadaghiani and Thiébaud
1987; Szabó et al. 2019). Trunk neural crest cells typically
migrate individually or in small clusters (Krotoski et al.
1988; Theveneau and Mayor 2012b; Vega-Lopez et al. 2017;
Shellard et al. 2018; Li, Vieceli et al. 2019).
In most vertebrates, neural crest migration requires a
“Cadherin switch,” in which pro-epithelial Cadherins, such
as Cdh1, are degraded and transcriptionally repressed and
replaced on the cell surface with Cadherins that enable
migration (e.g. Cdh6, Cdh7, Cdh11) (Levi et al. 1991; Vallin
et al. 1998; Nandadasa et al. 2009; Theveneau and Mayor
2012a; Langhe et al. 2016; Taneyhill and Schiffmacher
2017). Of these, Cdh11 is a target for cleavage by various
proteases, and one of its soluble cleavage products interacts with Cdgfra, Cgfr1, and Crbb2—all of which promote
migration (Mathavan et al. 2017). Interestingly, while cdh11
is highly expressed in migratory neural crest in Xenopus,
(Vallin et al. 1998; Borchers et al. 2001; Becker et al. 2013),
cdh1 is as well (Huang et al. 2016; Cousin 2017), raising
questions about the role for classical Cadherin switching in
neural crest migration in amphibians.
Neural crest migration requires active remodeling of the
extracellular environment, as well as interplay between the
extracellular environment, cell surface, nucleus, and cytoplasm. Xenopus is an advantageous system in which to dissect
these complex interactions, particularly given the ability to
carry out lateralized manipulations that use the contralateral
side of the same embryo as a control for normal migration
and to combine both in vivo and ex vivo experiments. A recent
fundamental discovery that took advantage of these attributes
showed the degree to which mechanical forces drive neural
crest behavior. This elegant study showed that the mesoderm
surrounding premigratory neural crest undergoes stiffening
and that the mechanical forces of this stiffening are both necessary and suffcient to trigger migration ( Barriga et al. 2018 ).
Integrin, Vinculin, and Talin proteins sense these mechanical
perturbations and then relay this information inside the cell
to mediate gene regulatory changes necessary for migration.
This work built on prior studies in Xenopus demonstrating
the importance of interactions between cell surface proteins
and the extracellular environment for controlling migration ( Epperlein et al. 1988 ). Many of these interactions also
require the activity of proteases, including Adam33, which
has been shown to remodel fbronectin substrates and cadherins ( Alfandari et al. 1997 ; Cai et al. 1998 ; McCusker et al.
2009 ; Cousin et al. 2012 ; Abbruzzese et al. 2016 ).
8.3.3. NEURAL CREST LINEAGE DIVERSIFICATION
One of the hallmarks of the neural crest is its capacity to generate remarkably diverse cell types and structures, including
cartilage; bone; neurons; glia; melanocytes; and components
of the endocrine system, heart, and teeth (Le Douarin and
Xenopus
across bilaterians. They are also instrumental in specifying a subset of neural crest cells. In Xenopus, morpholinomediated depletion of Twist1 leads to defects in neural crest
specifcation and migration and ultimately results in loss of
neural crest-derived elements of the head skeleton ( Lander
et al. 2011 ). Forced expression of Twist1 in Xenopus also
partially inhibits some aspects of neural crest development,
in part by pushing them toward an ecto-mesenchymal fate at
the expense of non-ectomesenchyme ( Lander et al. 2011 ).
Twist1, along with several other factors (Snai1, Sip1, Hif1a),
regulates EMTs in diverse cell types, including neural crest
in Xenopus ( Linker et al. 2000 ; Lander et al. 2011 ; Barriga
et al. 2013 ). Twist1 mediates this EMT program in part by
physically interacting with and modulating the activity of
Snai1/Snai2 proteins, which is regulated by Gsk3ß-mediated
phosphorylation ( Lander et al. 2013 ).
Snail family transcription factors are also important for
regulating cell migration across metazoans (Nieto 2002). In
most vertebrates, including Xenopus, both Snai1 and Snai2
factors are essential for regulating neural crest EMT (Carl,
Dufton et al. 1999; LaBonne and Bronner-Fraser 2000;
Aybar et al. 2003; Heeg-Truesdell and LaBonne 2004),
although there is evidence that some of their functions are
partially redundant and have been swapped during evolution (Locascio et al. 2002). The mechanisms by which Snai1
and Snai2 control neural crest EMT have been a topic of
intense study. This typically occurs by direct transcriptional
repression of genes that promote epithelial integrity (e.g.
cdh1) (Cano et al. 2000; Langer et al. 2008), which is often
enhanced by physical interaction with other EMT factors,
including the Polycomb repressor complex (Tien et al. 2015),
lmo4 (Ochoa et al. 2012; Ferronha et al. 2013), and Ajuba
LIM-domain proteins (Langer et al. 2008). Moreover, the
ability of Snai1 and Snai2 to exert their effects on neural
crest formation and migration are highly dependent upon
their stability, which is controlled by the F-box protein Ppa
(Vernon and LaBonne 2006; Lander et al. 2011) and can be
stabilized by Elp3 (Yang et al. 2016).
The forkhead box/winged helix transcription factor Foxd3
also plays critical roles in both controlling the neural crest
stem cell state and in promoting EMT. Work in Xenopus has
shown that knockdown of Foxd3 activity reduces expression of neural crest regulatory factors and causes defects in
neural crest migration (Sasai et al. 2001). A similar result
is obtained when foxd3 is ectopically expressed, suggesting
that precise levels of this transcription factor are required
for normal neural crest development (Pohl and Knöchel
2001). Although homologs of forkhead/fox genes are found
in invertebrates, the vertebrate foxd3 paralog is uniquely
suited for specifcation and EMT in the neural crest by having evolved a unique N-terminal sequence (Ono et al. 2014).
Because neural crest cells contribute derivatives throughout the body plan, they must migrate extensively to reach
those sites (Theveneau and Mayor 2012b; Gouignard et al.
2018 ). Xenopus is the only tetrapod model in which this complex process can be studied both in vitro and in vivo , enabling
fne-grained quantitative analysis of the migratory properties
of neural crest cells (Borchers et al. 2000; Alfandari et al.
2010; Toro-Tapia et al. 2017; Barriga et al. 2019). The cranial neural crest in amphibians initiates migration in three
discrete streams along the anterior-posterior axis, termed
mandibular, hyoid, and branchial (Sadaghiani and Thiébaud
1987; Szabó et al. 2019). Trunk neural crest cells typically
migrate individually or in small clusters (Krotoski et al.
1988; Theveneau and Mayor 2012b; Vega-Lopez et al. 2017;
Shellard et al. 2018; Li, Vieceli et al. 2019).
In most vertebrates, neural crest migration requires a
“Cadherin switch,” in which pro-epithelial Cadherins, such
as Cdh1, are degraded and transcriptionally repressed and
replaced on the cell surface with Cadherins that enable
migration (e.g. Cdh6, Cdh7, Cdh11) (Levi et al. 1991; Vallin
et al. 1998; Nandadasa et al. 2009; Theveneau and Mayor
2012a; Langhe et al. 2016; Taneyhill and Schiffmacher
2017). Of these, Cdh11 is a target for cleavage by various
proteases, and one of its soluble cleavage products interacts with Cdgfra, Cgfr1, and Crbb2—all of which promote
migration (Mathavan et al. 2017). Interestingly, while cdh11
is highly expressed in migratory neural crest in Xenopus,
(Vallin et al. 1998; Borchers et al. 2001; Becker et al. 2013),
cdh1 is as well (Huang et al. 2016; Cousin 2017), raising
questions about the role for classical Cadherin switching in
neural crest migration in amphibians.
Neural crest migration requires active remodeling of the
extracellular environment, as well as interplay between the
extracellular environment, cell surface, nucleus, and cytoplasm. Xenopus is an advantageous system in which to dissect
these complex interactions, particularly given the ability to
carry out lateralized manipulations that use the contralateral
side of the same embryo as a control for normal migration
and to combine both in vivo and ex vivo experiments. A recent
fundamental discovery that took advantage of these attributes
showed the degree to which mechanical forces drive neural
crest behavior. This elegant study showed that the mesoderm
surrounding premigratory neural crest undergoes stiffening
and that the mechanical forces of this stiffening are both necessary and suffcient to trigger migration ( Barriga et al. 2018 ).
Integrin, Vinculin, and Talin proteins sense these mechanical
perturbations and then relay this information inside the cell
to mediate gene regulatory changes necessary for migration.
This work built on prior studies in Xenopus demonstrating
the importance of interactions between cell surface proteins
and the extracellular environment for controlling migration ( Epperlein et al. 1988 ). Many of these interactions also
require the activity of proteases, including Adam33, which
has been shown to remodel fbronectin substrates and cadherins ( Alfandari et al. 1997 ; Cai et al. 1998 ; McCusker et al.
2009 ; Cousin et al. 2012 ; Abbruzzese et al. 2016 ).
8.3.3. NEURAL CREST LINEAGE DIVERSIFICATION
One of the hallmarks of the neural crest is its capacity to generate remarkably diverse cell types and structures, including
cartilage; bone; neurons; glia; melanocytes; and components
of the endocrine system, heart, and teeth (Le Douarin and