238
Xenopus
disturbed, it may reveal unexpected roles for these genes.
One such well-studied family is the Nucleoporins (Nups).
Nups are proteins that make up the approximately 100
Megadalton (MDa) nuclear pore complexes (NPCs), which
are the gatekeepers for transport in and out of the nucleus
(Viso et al. 2016). The PCGC identif ed patient mutations
in several Nups (Jin et al. 2017), found in various NPC substructures. However, since nuclear transport is fundamental
for all cells, how can altering it be associated with CHD/
HTX, as opposed to organism survival?
The frst CHD/HTX candidate Nup studied in depth for
its role in early development is Nup188, which was identif ed
via CNV analysis (Fakhro et al. 2011). The investigators f rst
explored the role of Nup188 in organ situs. Its knockdown
via translation-blocking morpholino led to abnormal heart
looping, gut looping, and pitx2c expression (Fakhro et al.
2011). However, how does Nup188 regulate LR patterning?
Nup188 is a component of the inner ring of the nuclear pore
complex and binds directly to Nup93. Therefore, the investigators depleted Nup93 to see if it recapitulated the phenotypes in Nup188 depletion, and indeed, it did (Viso et al.
2016). This appeared specif c to the inner ring, as depletion
of components of the outer ring and the central transport
channel did not recapitulate Nup188 depletion phenotypes.
Thus, the inner ring Nups specifcally play a role in heart
looping.
To look more deeply at this mechanism, the authors made
a surprising observation. Embryos depleted of Nup93 and
Nup188 did not glide along the surface of a Petri dish, unlike
their wild-type siblings. Multiciliated cells on the embryonic epidermis beat and create extracellular f uid f ow that
drives this gliding. By immunohistochemistry, most cilia
in these cells were depleted. This was also true of the cilia
in the LRO. The authors therefore demonstrated that innerring Nup188 and Nup93 are uniquely important for cilia
structure, as their depletion led to dramatic changes in cilia
structure. However, gross nuclear pore number, structure,
and function appeared intact. While the authors could not
eliminate the possibility of a subtle defect, the authors began
to look for mechanisms of how inner-ring Nups might affect
cilia structure independently of nucleocytoplasmic transport
and NPC structure.
If inner-ring Nups do not affect cilia structure via nucleocytoplasmic transport, what else could they affect? Previous
studies showed that NPC substructures can have functions
outside of the NPC (Belgareh et al. 2001; Loïodice et al.
2004; Hashizume et al. 2013; Itoh et al. 2013). One study
proposed a model for a “ciliary pore complex” similar in
function to the NPC that provides a size-selective barrier
for entry of proteins into the ciliary axoneme (Kee et al.
2012). However, the evidence supporting this model has
been disputed (Breslow et al. 2013). With this in mind, the
authors investigated whether inner-ring Nups localize at the
base of cilia. Immunofuorescence imaging of endogenous
Nup93 and Nup188 in retinal pigmented epithelial (RPE)
cells revealed their accumulation near the basal-bodies,
while other non-inner ring Nups did not. This suggests that
Nup188 and Nup93 have a role independent of the NPC at
cilia and that NPC components normally needed to create
the NPC diffusion barrier do not localize at the base of cilia,
contradicting the “ciliary pore complex” model.
To visualize the structure of the inner-ring Nups at the
base of cilia, the authors used super-resolution microscopy
to visualize Nup93 and Nup188 structures at the ciliary
base. Nup188 and Nup93 clustered in much smaller puncta
(about 50 nm), contradicting the 100-nm ring prediction for
a ciliary pore. In fact, these small clusters distributed in a
pattern around the basal bodies in two barrel-like structures, a pattern congruent with the pericentriolar material
(PCM) surrounding mother and daughter centrioles (Viso
et al. 2016). This surprising discovery indicated that depletion of Nup188 contributes to CHD/HTX by disrupting the
formation of cilia needed to establish proper LR patterning
through a novel role in ciliary and centrosomal biology. This
study that began with a CHD patient opened a new avenue
to explore connections between Nups, centrioles, and cilia
(Vishnoi et al. 2020).
16.4.3. EXPLORING OLD MYSTERIES IN
DEVELOPMENTAL CELL SIGNALING
Linking CHD/HTX variants to a developmental phenotype
is often the starting point for achieving a better understanding of basic biological questions. Sometimes these questions
can lead to a novel perspective on long-standing questions,
for example, the nuclear transport of β-Catenin, a key effector of the Wnt signaling pathway. During Wnt signaling,
Wnt ligand binding leads to sequestration of the β-Catenin
degradation complex so that β-Catenin is stabilized and
can accumulate and enter the nucleus to activate Wnt target
genes. In fact, the tight regulation of β-Catenin is essential,
and its overexpression contributes to context-dependent diseases: in the context of the early embryo, excessive ectopic
ventral Wnt signaling can lead to a double axis (a conjoinedtwin embryo) and in adult cells to cancers.
β-Catenin uses facilitated transport to enter the nucleus
independent of Importin α/β1. Remarkably, previous studies
indicated that β-Catenin nuclear transport was independent
of Ran, the master regulator of facilitated nuclear transport
that employs nuclear transport receptors (Fagotto, Glück,
and Gumbiner 1998; Yokoya et al. 1999). However, nuclear
transport was energy dependent, which led to the hypothesis
that either (1) β-Catenin binds directly to the NPC via several Armadillo (ARM) repeats and acts as its own nuclear
transport receptor (NTR) (Fagotto, Glück, and Gumbiner
1998), or (2) β-Catenin employs another GTPase that is not
Ran. However, evidence for a direct interaction between
β-Catenin and the NPC is disputed (Sharma et al. 2014; Suh
and Gumbiner 2003). While the negative data mounted, a
fundamental question with major impact for a host of Wntrelated diseases remained: How does β-Catenin enter the
nucleus?
A guanine nuclear exchange factor, RAPGEF5, was
identifed as a CHD/HTX candidate (Fakhro et al. 2011),
Xenopus
disturbed, it may reveal unexpected roles for these genes.
One such well-studied family is the Nucleoporins (Nups).
Nups are proteins that make up the approximately 100
Megadalton (MDa) nuclear pore complexes (NPCs), which
are the gatekeepers for transport in and out of the nucleus
(Viso et al. 2016). The PCGC identif ed patient mutations
in several Nups (Jin et al. 2017), found in various NPC substructures. However, since nuclear transport is fundamental
for all cells, how can altering it be associated with CHD/
HTX, as opposed to organism survival?
The frst CHD/HTX candidate Nup studied in depth for
its role in early development is Nup188, which was identif ed
via CNV analysis (Fakhro et al. 2011). The investigators f rst
explored the role of Nup188 in organ situs. Its knockdown
via translation-blocking morpholino led to abnormal heart
looping, gut looping, and pitx2c expression (Fakhro et al.
2011). However, how does Nup188 regulate LR patterning?
Nup188 is a component of the inner ring of the nuclear pore
complex and binds directly to Nup93. Therefore, the investigators depleted Nup93 to see if it recapitulated the phenotypes in Nup188 depletion, and indeed, it did (Viso et al.
2016). This appeared specif c to the inner ring, as depletion
of components of the outer ring and the central transport
channel did not recapitulate Nup188 depletion phenotypes.
Thus, the inner ring Nups specifcally play a role in heart
looping.
To look more deeply at this mechanism, the authors made
a surprising observation. Embryos depleted of Nup93 and
Nup188 did not glide along the surface of a Petri dish, unlike
their wild-type siblings. Multiciliated cells on the embryonic epidermis beat and create extracellular f uid f ow that
drives this gliding. By immunohistochemistry, most cilia
in these cells were depleted. This was also true of the cilia
in the LRO. The authors therefore demonstrated that innerring Nup188 and Nup93 are uniquely important for cilia
structure, as their depletion led to dramatic changes in cilia
structure. However, gross nuclear pore number, structure,
and function appeared intact. While the authors could not
eliminate the possibility of a subtle defect, the authors began
to look for mechanisms of how inner-ring Nups might affect
cilia structure independently of nucleocytoplasmic transport
and NPC structure.
If inner-ring Nups do not affect cilia structure via nucleocytoplasmic transport, what else could they affect? Previous
studies showed that NPC substructures can have functions
outside of the NPC (Belgareh et al. 2001; Loïodice et al.
2004; Hashizume et al. 2013; Itoh et al. 2013). One study
proposed a model for a “ciliary pore complex” similar in
function to the NPC that provides a size-selective barrier
for entry of proteins into the ciliary axoneme (Kee et al.
2012). However, the evidence supporting this model has
been disputed (Breslow et al. 2013). With this in mind, the
authors investigated whether inner-ring Nups localize at the
base of cilia. Immunofuorescence imaging of endogenous
Nup93 and Nup188 in retinal pigmented epithelial (RPE)
cells revealed their accumulation near the basal-bodies,
while other non-inner ring Nups did not. This suggests that
Nup188 and Nup93 have a role independent of the NPC at
cilia and that NPC components normally needed to create
the NPC diffusion barrier do not localize at the base of cilia,
contradicting the “ciliary pore complex” model.
To visualize the structure of the inner-ring Nups at the
base of cilia, the authors used super-resolution microscopy
to visualize Nup93 and Nup188 structures at the ciliary
base. Nup188 and Nup93 clustered in much smaller puncta
(about 50 nm), contradicting the 100-nm ring prediction for
a ciliary pore. In fact, these small clusters distributed in a
pattern around the basal bodies in two barrel-like structures, a pattern congruent with the pericentriolar material
(PCM) surrounding mother and daughter centrioles (Viso
et al. 2016). This surprising discovery indicated that depletion of Nup188 contributes to CHD/HTX by disrupting the
formation of cilia needed to establish proper LR patterning
through a novel role in ciliary and centrosomal biology. This
study that began with a CHD patient opened a new avenue
to explore connections between Nups, centrioles, and cilia
(Vishnoi et al. 2020).
16.4.3. EXPLORING OLD MYSTERIES IN
DEVELOPMENTAL CELL SIGNALING
Linking CHD/HTX variants to a developmental phenotype
is often the starting point for achieving a better understanding of basic biological questions. Sometimes these questions
can lead to a novel perspective on long-standing questions,
for example, the nuclear transport of β-Catenin, a key effector of the Wnt signaling pathway. During Wnt signaling,
Wnt ligand binding leads to sequestration of the β-Catenin
degradation complex so that β-Catenin is stabilized and
can accumulate and enter the nucleus to activate Wnt target
genes. In fact, the tight regulation of β-Catenin is essential,
and its overexpression contributes to context-dependent diseases: in the context of the early embryo, excessive ectopic
ventral Wnt signaling can lead to a double axis (a conjoinedtwin embryo) and in adult cells to cancers.
β-Catenin uses facilitated transport to enter the nucleus
independent of Importin α/β1. Remarkably, previous studies
indicated that β-Catenin nuclear transport was independent
of Ran, the master regulator of facilitated nuclear transport
that employs nuclear transport receptors (Fagotto, Glück,
and Gumbiner 1998; Yokoya et al. 1999). However, nuclear
transport was energy dependent, which led to the hypothesis
that either (1) β-Catenin binds directly to the NPC via several Armadillo (ARM) repeats and acts as its own nuclear
transport receptor (NTR) (Fagotto, Glück, and Gumbiner
1998), or (2) β-Catenin employs another GTPase that is not
Ran. However, evidence for a direct interaction between
β-Catenin and the NPC is disputed (Sharma et al. 2014; Suh
and Gumbiner 2003). While the negative data mounted, a
fundamental question with major impact for a host of Wntrelated diseases remained: How does β-Catenin enter the
nucleus?
A guanine nuclear exchange factor, RAPGEF5, was
identifed as a CHD/HTX candidate (Fakhro et al. 2011),