237
Discovering the Function of CHD Genes
tissue patterning and ciliogenesis ( Zhang et al. 2012) and
pitx2c expression in the cardiac neural crest (Kioussi et al.
2002). Non-canonical Wnt/planar cell polarity (PCP) pathways contribute to cilia polarization in the LRO (Minegishi
et al. 2017). Notch signaling establishes the immotile:motile
cilia ratio in the LRO (Boskovski et al. 2013) and is important for nodal expression (Raya et al. 2003; Krebs et al.
2003). BMP signaling regulates right-side identity establishment (Ocaña et al. 2017), whereas Nodal signaling is important for left-side identity establishment (Schweickert et al.
2010; Desgrange, Le Garrec, and Meilhac 2018). Disruption
of the cellular mechanisms underlying any of these pathways could result in disastrous LR patterning and subsequent disruptions in visceral organ and cardiac structure and
organization.
16.4. PATIENT-DERIVED CHD DISEASE
MECHANISMS UNCOVERED IN XENOPUS
Researchers have used the strategies described previously to
identify and study the roles of CHD candidate genes in early
embryonic development. This section will highlight several
studies of the roles of several CHD candidate genes.
16.4.1. CONNECTING KNOWN PATHWAYS IN A NEW
CONTEXT: GLYCOSYLATION IN NOTCH
SIGNALING AND CILIA CELL FATE DETERMINATION
CHD candidate genes may be involved in well-def ned pathways, such as Notch signaling or glycosylation. However,
the role of these pathways in early embryonic development
and CHD/HTX is often unexplored or poorly understood.
One such CHD candidate, galnt11, was identif ed as
a CNV deletion in a HTX patient (Fakhro et al. 2011).
galnt11 is a member of the protein family necessary for
the O-glycosylation of N-acetylgalactosamines (GalNAc)
(Bennett et al. 2012). The role of Galnt11 or GalNAc
O-glycosylation had not yet been studied in the context
of embryonic development, and at the time, no targets of
galnt11 were known. In order to investigate its role in
CHD/HTX, investigators depleted galnt11 in X. tropicalis
embryos. These embryos had abnormal gut and heart looping (Fakhro et al. 2011; Boskovski et al. 2013), indicating
galnt11’s role in LR organ situs. Investigating earlier steps in
the LR-pathway revealed that both pitx2c and dand5 expression were abnormal, suggesting a defect in cilia at the LRO.
As a proxy to cilia in the LRO, the authors investigated
the multiciliated cells of the embryonic epidermis. They did
not fnd any abnormalities in the cilia structure in these cells.
However, careful inspection revealed a dramatic increase in
number of multiciliated cells after galnt11 depletion. In contrast, overexpressing galnt11 reduced the number of multiciliated cells. This phenotype was also seen when the Notch
pathway was manipulated (Liu et al. 2007; Ma and Jiang
2007; Tsao et al. 2009). Thus, the authors proposed that
galnt11 may work through Notch signaling to affect multiciliated cell number.
In Notch signaling, extracellular ligands, Delta or Jagged,
bind to the Notch receptor, causing physical changes to the
receptor that make it amenable to extracellular cleavage by
the ADAM metalloprotease; this initiates an intracellular
cleavage by Ɣ-secretase and releases the Notch Intracellular
Domain (NICD). NICD then translocates into the nucleus
and binds to target genes via an interaction with the CSL
transcription complex. NICD and a constitutively active CSL,
but not Delta, rescued LR phenotypes in galnt11 -depleted
embryos. This suggested that galnt11 impacts LR patterning
through the Notch pathway at a step between binding of the
extracellular ligand and NICD release into the cytoplasm.
Could the Notch receptor be a target of Galnt11? Previous
studies had demonstrated that Notch is glycosylated, but not by
a GalNac type glycosylation enzyme. The authors used mass
spectrometry on the Notch extracellular domain peptides and
an in vitro glycosylation assay to identify potential GalNac
O-glycosylation sites. One of the three identifed target sites
is adjacent to the extracellular ADAM cleavage site. In previous work, GalNac glycosylation near a cleavage site generally
prevented cleavage of the pro-peptide. However, in this case,
Galnt11 appeared to enhance Notch signaling (loss of Galnt11
mimicked a loss of Notch phenotype, and gain of Notch rescued loss of galnt11 phenotypes). Unlike previous studies, in
the case of the Notch receptor, Galnt11 glycosylation enhanced
cleavage. This supports the interpretation that Galnt11
O-glycosylation of this adjacent site is important for proper
Notch receptor processing, signaling, and LR patterning.
An important question is: How does Galnt11 and Notch
signaling impact LR patterning? Previously, a two-cilia
model had been proposed for vertebrate LR axis determination (McGrath et al. 2003; Tabin and Vogan 2003). The
model proposed that one class of cilia in the LRO are responsible for generating fow, while another is required for sensing and translating that fow-based signal. However, how the
LRO establishes both motile and immotile cilia was simply
unknown. Because galnt11/Notch loss-of-function produced
phenotypes that suggested a loss of cilia sensing, whereas
galnt11/Notch gain-of-function led to phenotypes suggesting loss of cilia motility, the authors hypothesized that Notch
may act as a switch between motile and immotile cilia types,
which could solve a long-standing mystery in the two-cilia
model. Using live cell imaging, the authors found that Notch/
Galnt11 signaling altered the balance between motile and
immotile cilia. Ultimately, the authors not only identif ed the
role of galnt11 in CHD/HTX pathogenesis but also uncovered a previously unknown pathway for regulating Notch
signaling and the subsequent cilia distribution required for
regulation of the LR patterning (Boskovski et al. 2013).
16.4.2. UNCOVERING NOVEL ROLES FOR WELLSTUDIED GENES: NUCLEOPORINS IN
CILIA AND CENTROSOMAL BIOLOGY
Some CHD candidate genes have well-studied cellular functions that researchers may not have looked at in the context of early embryonic development. When development is
Discovering the Function of CHD Genes
tissue patterning and ciliogenesis ( Zhang et al. 2012) and
pitx2c expression in the cardiac neural crest (Kioussi et al.
2002). Non-canonical Wnt/planar cell polarity (PCP) pathways contribute to cilia polarization in the LRO (Minegishi
et al. 2017). Notch signaling establishes the immotile:motile
cilia ratio in the LRO (Boskovski et al. 2013) and is important for nodal expression (Raya et al. 2003; Krebs et al.
2003). BMP signaling regulates right-side identity establishment (Ocaña et al. 2017), whereas Nodal signaling is important for left-side identity establishment (Schweickert et al.
2010; Desgrange, Le Garrec, and Meilhac 2018). Disruption
of the cellular mechanisms underlying any of these pathways could result in disastrous LR patterning and subsequent disruptions in visceral organ and cardiac structure and
organization.
16.4. PATIENT-DERIVED CHD DISEASE
MECHANISMS UNCOVERED IN XENOPUS
Researchers have used the strategies described previously to
identify and study the roles of CHD candidate genes in early
embryonic development. This section will highlight several
studies of the roles of several CHD candidate genes.
16.4.1. CONNECTING KNOWN PATHWAYS IN A NEW
CONTEXT: GLYCOSYLATION IN NOTCH
SIGNALING AND CILIA CELL FATE DETERMINATION
CHD candidate genes may be involved in well-def ned pathways, such as Notch signaling or glycosylation. However,
the role of these pathways in early embryonic development
and CHD/HTX is often unexplored or poorly understood.
One such CHD candidate, galnt11, was identif ed as
a CNV deletion in a HTX patient (Fakhro et al. 2011).
galnt11 is a member of the protein family necessary for
the O-glycosylation of N-acetylgalactosamines (GalNAc)
(Bennett et al. 2012). The role of Galnt11 or GalNAc
O-glycosylation had not yet been studied in the context
of embryonic development, and at the time, no targets of
galnt11 were known. In order to investigate its role in
CHD/HTX, investigators depleted galnt11 in X. tropicalis
embryos. These embryos had abnormal gut and heart looping (Fakhro et al. 2011; Boskovski et al. 2013), indicating
galnt11’s role in LR organ situs. Investigating earlier steps in
the LR-pathway revealed that both pitx2c and dand5 expression were abnormal, suggesting a defect in cilia at the LRO.
As a proxy to cilia in the LRO, the authors investigated
the multiciliated cells of the embryonic epidermis. They did
not fnd any abnormalities in the cilia structure in these cells.
However, careful inspection revealed a dramatic increase in
number of multiciliated cells after galnt11 depletion. In contrast, overexpressing galnt11 reduced the number of multiciliated cells. This phenotype was also seen when the Notch
pathway was manipulated (Liu et al. 2007; Ma and Jiang
2007; Tsao et al. 2009). Thus, the authors proposed that
galnt11 may work through Notch signaling to affect multiciliated cell number.
In Notch signaling, extracellular ligands, Delta or Jagged,
bind to the Notch receptor, causing physical changes to the
receptor that make it amenable to extracellular cleavage by
the ADAM metalloprotease; this initiates an intracellular
cleavage by Ɣ-secretase and releases the Notch Intracellular
Domain (NICD). NICD then translocates into the nucleus
and binds to target genes via an interaction with the CSL
transcription complex. NICD and a constitutively active CSL,
but not Delta, rescued LR phenotypes in galnt11 -depleted
embryos. This suggested that galnt11 impacts LR patterning
through the Notch pathway at a step between binding of the
extracellular ligand and NICD release into the cytoplasm.
Could the Notch receptor be a target of Galnt11? Previous
studies had demonstrated that Notch is glycosylated, but not by
a GalNac type glycosylation enzyme. The authors used mass
spectrometry on the Notch extracellular domain peptides and
an in vitro glycosylation assay to identify potential GalNac
O-glycosylation sites. One of the three identifed target sites
is adjacent to the extracellular ADAM cleavage site. In previous work, GalNac glycosylation near a cleavage site generally
prevented cleavage of the pro-peptide. However, in this case,
Galnt11 appeared to enhance Notch signaling (loss of Galnt11
mimicked a loss of Notch phenotype, and gain of Notch rescued loss of galnt11 phenotypes). Unlike previous studies, in
the case of the Notch receptor, Galnt11 glycosylation enhanced
cleavage. This supports the interpretation that Galnt11
O-glycosylation of this adjacent site is important for proper
Notch receptor processing, signaling, and LR patterning.
An important question is: How does Galnt11 and Notch
signaling impact LR patterning? Previously, a two-cilia
model had been proposed for vertebrate LR axis determination (McGrath et al. 2003; Tabin and Vogan 2003). The
model proposed that one class of cilia in the LRO are responsible for generating fow, while another is required for sensing and translating that fow-based signal. However, how the
LRO establishes both motile and immotile cilia was simply
unknown. Because galnt11/Notch loss-of-function produced
phenotypes that suggested a loss of cilia sensing, whereas
galnt11/Notch gain-of-function led to phenotypes suggesting loss of cilia motility, the authors hypothesized that Notch
may act as a switch between motile and immotile cilia types,
which could solve a long-standing mystery in the two-cilia
model. Using live cell imaging, the authors found that Notch/
Galnt11 signaling altered the balance between motile and
immotile cilia. Ultimately, the authors not only identif ed the
role of galnt11 in CHD/HTX pathogenesis but also uncovered a previously unknown pathway for regulating Notch
signaling and the subsequent cilia distribution required for
regulation of the LR patterning (Boskovski et al. 2013).
16.4.2. UNCOVERING NOVEL ROLES FOR WELLSTUDIED GENES: NUCLEOPORINS IN
CILIA AND CENTROSOMAL BIOLOGY
Some CHD candidate genes have well-studied cellular functions that researchers may not have looked at in the context of early embryonic development. When development is