249
Craniofacial Development and Disorders
ectoderm and endoderm disappears, requiring activation of
the ß-catenin WNT antagonist frzb1 that is expressed in the
EAD (Dickinson and Sive 2009; Tabler et al. 2014). frzb1
expression in turn requires SHH expression (Tabler et al.
2014). At this stage, the stomodeum forms, as described in
all vertebrates (Waterman 1977; Waterman and Schoenwolf
1980; Som and Naidich 2013). The cell layers thin through
cell death and migration, until a single layer of ectoderm
and endoderm remains, the buccopharyngeal membrane
(BPM) (Dickinson and Sive 2006; Jacox et al. 2016), which
perforates, leading to the mouth opening (Figure 17.2A)
(Dickinson and Sive 2006). Bucopharyngeal membrane
perforation is mediated by JNK signaling acting through
ß-catenin phosphorylation and E-cadherin endocytosis at
adherens junctions (Houssin et al. 2017).
17.5. THE EXTREME ANTERIOR DOMAIN IS A
CRANIOFACIAL SIGNALING CENTER
17.5.1. IDENTIFICATION OF THE EXTREME ANTERIOR DOMAIN
Work from our group identif ed the Xenopus EAD as a craniofacial organizer (Jacox, Sindelka et al. 2014). The EAD
comprises approximately 500 cells at the anterior of the
embryo and is present from the late neurula through tailbud
stages. As noted, the EAD comprises directly juxtaposed
ectoderm and endoderm without the intervening mesoderm.
This type of tissue arrangement has been observed across
deuterostomes and is present in mammals (Chen, Jacox et al.
2017; Chen 2018; Soukup, Horacek, and Cerny 2013). The
EAD contributes to similar structures in multiple species,
including birds (Couly, Coltey, and Le Douarin 1993; Couly
and Le Douarin 1987), mice (Osumi-Yamashita et al. 1994),
zebrafsh (Eberhart et al. 2006; Chapman et al. 2005), and
frog (Eagleson, Ferreiro, and Harris 1995; Dickinson and
Sive 2006), which include the mouth, nostrils, and anterior
pituitary. Although this region is present in all chordates,
including humans, work from our group in Xenopus has provided pivotal functional analysis.
17.5.2. CRANIOFACIAL ORGANIZER FUNCTION
OF THE EXTREME ANTERIOR DOMAIN
Our group demonstrated that the Xenopus EAD has organizer function using a facial transplant protocol (Dickinson
and Sive 2009; Jacox, Dickinson, and Sive 2014; Jacox,
Sindelka et al. 2014). In particular, we observed that after
transplantation of an EAD that lacked function of the WNT
antagonists frzb1 and crescent into a control embryo, not
only did the mouth fail to form, but the rest of the face was
thin and undeveloped (Dickinson and Sive 2009). We further demonstrated that the EAD acted on frst arch migratory neural crest to promote its ingress into the developing
face ( Figure 17.2B) (Jacox, Sindelka et al. 2014). This inf uence required the kinin-kallikrein signaling system, shown
for the frst time to be involved in early craniofacial development (Jacox, Sindelka et al. 2014). The pathway includes
Kininogen encoded by the kng gene, a precursor for the
signaling peptide Bradykinin (Bdk), and the Bdk processing enzyme Carboxypeptidase N, encoded by the cpn gene.
cpn RNA is localized in the EAD, while kng RNA is more
broadly distributed. Kinin-kallikrein signaling culminates
in nitric oxide (NO) production, synthesized by Nitric Oxide
Synthase. We showed that NO was produced around the
EAD, was absent after local cpn LOF, and was enhanced
after implantation of beads coated with BDK peptide. This
study demonstrated that the EAD signals to the neural crest
through kinin-kallikrein pathway signaling to guide its
development. As noted in Section 4.3, in older embryos, the
f rst arch crest signals back to the EAD to promote its convergent extension and formation of a pre-mouth array that
later opens into the stomodeum (Figure 17.2B) (Jacox, Chen
et al. 2016). Chen (2018) demonstrated that the secreted
WNT antagonist fzb1 that is expressed locally in the EAD
also infuences neural crest development, including promoting proliferation, and importantly, also impacts brain development. These data demonstrate a global signaling role and
organizer function for EAD during craniofacial development. EAD perturbation would be associated with mouth
anomalies, and due to its signaling role, may contribute to
other craniofacial anomalies.
17.6. LANDSCAPE OF CRANIOFACIAL
ANOMALIES
Craniofacial disorders or anomalies occur during formation
of the skull and facial bones and are generally congenital or
present at birth. Some CFA are syndromic, associated with
def ned genetic changes (Buchanan, Xue, and Hollier 2014;
Rice 2005; Nagy and Demke 2014).
17.6.1. CRANIOFACIAL ANOMALIES ARE
ASSOCIATED WITH MULTIPLE FACTORS
The causes of CFA are often unknown or complex but are
considered to include the following.
1. Genetic. Gene variants associated with CFA include
genes that regulate DNA replication, cell proliferation, cell signaling, or transcription (Roosenboom
et al. 2016; Kobayashi et al. 2013; Alappat, Zhang,
and Chen 2003; Gebuijs et al. 2019; Merkuri and
Fish 2019; Twigg and Wilkie 2015; Bartzela,
Carels, and Maltha 2017).
2. Environmental. Medication, recreational drugs,
and alcohol during pregnancy have also been linked
with certain craniofacial anomalies (Thompson,
Levitt, and Stanwood 2009; Moiseiwitsch 2000;
Seda et al. 2019; Muggli et al. 2017; Sulik 2005).
Similarly, exposure to viruses such as Zika (Yan
et al. 2019; Wheeler 2018, 2020) and cytomegalovirus (CMV) (Cheeran, Lokensgard, and Schleiss
2009; Jaskoll et al. 2008; Weichert et al. 2010)
Craniofacial Development and Disorders
ectoderm and endoderm disappears, requiring activation of
the ß-catenin WNT antagonist frzb1 that is expressed in the
EAD (Dickinson and Sive 2009; Tabler et al. 2014). frzb1
expression in turn requires SHH expression (Tabler et al.
2014). At this stage, the stomodeum forms, as described in
all vertebrates (Waterman 1977; Waterman and Schoenwolf
1980; Som and Naidich 2013). The cell layers thin through
cell death and migration, until a single layer of ectoderm
and endoderm remains, the buccopharyngeal membrane
(BPM) (Dickinson and Sive 2006; Jacox et al. 2016), which
perforates, leading to the mouth opening (Figure 17.2A)
(Dickinson and Sive 2006). Bucopharyngeal membrane
perforation is mediated by JNK signaling acting through
ß-catenin phosphorylation and E-cadherin endocytosis at
adherens junctions (Houssin et al. 2017).
17.5. THE EXTREME ANTERIOR DOMAIN IS A
CRANIOFACIAL SIGNALING CENTER
17.5.1. IDENTIFICATION OF THE EXTREME ANTERIOR DOMAIN
Work from our group identif ed the Xenopus EAD as a craniofacial organizer (Jacox, Sindelka et al. 2014). The EAD
comprises approximately 500 cells at the anterior of the
embryo and is present from the late neurula through tailbud
stages. As noted, the EAD comprises directly juxtaposed
ectoderm and endoderm without the intervening mesoderm.
This type of tissue arrangement has been observed across
deuterostomes and is present in mammals (Chen, Jacox et al.
2017; Chen 2018; Soukup, Horacek, and Cerny 2013). The
EAD contributes to similar structures in multiple species,
including birds (Couly, Coltey, and Le Douarin 1993; Couly
and Le Douarin 1987), mice (Osumi-Yamashita et al. 1994),
zebrafsh (Eberhart et al. 2006; Chapman et al. 2005), and
frog (Eagleson, Ferreiro, and Harris 1995; Dickinson and
Sive 2006), which include the mouth, nostrils, and anterior
pituitary. Although this region is present in all chordates,
including humans, work from our group in Xenopus has provided pivotal functional analysis.
17.5.2. CRANIOFACIAL ORGANIZER FUNCTION
OF THE EXTREME ANTERIOR DOMAIN
Our group demonstrated that the Xenopus EAD has organizer function using a facial transplant protocol (Dickinson
and Sive 2009; Jacox, Dickinson, and Sive 2014; Jacox,
Sindelka et al. 2014). In particular, we observed that after
transplantation of an EAD that lacked function of the WNT
antagonists frzb1 and crescent into a control embryo, not
only did the mouth fail to form, but the rest of the face was
thin and undeveloped (Dickinson and Sive 2009). We further demonstrated that the EAD acted on frst arch migratory neural crest to promote its ingress into the developing
face ( Figure 17.2B) (Jacox, Sindelka et al. 2014). This inf uence required the kinin-kallikrein signaling system, shown
for the frst time to be involved in early craniofacial development (Jacox, Sindelka et al. 2014). The pathway includes
Kininogen encoded by the kng gene, a precursor for the
signaling peptide Bradykinin (Bdk), and the Bdk processing enzyme Carboxypeptidase N, encoded by the cpn gene.
cpn RNA is localized in the EAD, while kng RNA is more
broadly distributed. Kinin-kallikrein signaling culminates
in nitric oxide (NO) production, synthesized by Nitric Oxide
Synthase. We showed that NO was produced around the
EAD, was absent after local cpn LOF, and was enhanced
after implantation of beads coated with BDK peptide. This
study demonstrated that the EAD signals to the neural crest
through kinin-kallikrein pathway signaling to guide its
development. As noted in Section 4.3, in older embryos, the
f rst arch crest signals back to the EAD to promote its convergent extension and formation of a pre-mouth array that
later opens into the stomodeum (Figure 17.2B) (Jacox, Chen
et al. 2016). Chen (2018) demonstrated that the secreted
WNT antagonist fzb1 that is expressed locally in the EAD
also infuences neural crest development, including promoting proliferation, and importantly, also impacts brain development. These data demonstrate a global signaling role and
organizer function for EAD during craniofacial development. EAD perturbation would be associated with mouth
anomalies, and due to its signaling role, may contribute to
other craniofacial anomalies.
17.6. LANDSCAPE OF CRANIOFACIAL
ANOMALIES
Craniofacial disorders or anomalies occur during formation
of the skull and facial bones and are generally congenital or
present at birth. Some CFA are syndromic, associated with
def ned genetic changes (Buchanan, Xue, and Hollier 2014;
Rice 2005; Nagy and Demke 2014).
17.6.1. CRANIOFACIAL ANOMALIES ARE
ASSOCIATED WITH MULTIPLE FACTORS
The causes of CFA are often unknown or complex but are
considered to include the following.
1. Genetic. Gene variants associated with CFA include
genes that regulate DNA replication, cell proliferation, cell signaling, or transcription (Roosenboom
et al. 2016; Kobayashi et al. 2013; Alappat, Zhang,
and Chen 2003; Gebuijs et al. 2019; Merkuri and
Fish 2019; Twigg and Wilkie 2015; Bartzela,
Carels, and Maltha 2017).
2. Environmental. Medication, recreational drugs,
and alcohol during pregnancy have also been linked
with certain craniofacial anomalies (Thompson,
Levitt, and Stanwood 2009; Moiseiwitsch 2000;
Seda et al. 2019; Muggli et al. 2017; Sulik 2005).
Similarly, exposure to viruses such as Zika (Yan
et al. 2019; Wheeler 2018, 2020) and cytomegalovirus (CMV) (Cheeran, Lokensgard, and Schleiss
2009; Jaskoll et al. 2008; Weichert et al. 2010)