239
Discovering the Function of CHD Genes
but no role for it in embryonic development was known
( Griffn et al. 2018). Depleting rapgef5 in X. tropicalis
embryos led to abnormal cardiac looping similar to the
patient phenotype, suggesting a role in early development.
Indeed, rapgef5 depletion also affected earlier markers
of LR patterning, pitx2 and dand5. However, unlike the
two previous examples of CHD candidate genes, rapgef5
depletion reduced dand5 expression even before the commencement of cilia-driven fow. This suggested that rapgef5 depletion affected the specifcation of the LRO prior
to cilia-driven signaling.
The LRO is a transient mesodermal structure that is
overlaid by the endoderm as gastrulation is completed.
Therefore, any defects in gastrulation or formation of the
mesoderm can affect the formation of the LRO and LR signaling. As depletion of rapgef5 affected the formation of the
LRO, investigators examined other markers of the mesoderm and found that most were normal, except the known
Wnt responsive genes foxj1 and nodal3.1. These results indicated that rapgef5 likely disrupts LRO patterning and subsequent LR patterning by impacting Wnt signaling. Consistent
with this f nding, rapgef5 depletion reduced embryonic
β-Catenin levels. Given the many genes involved in transducing the Wnt signal into the cell and the degradation of
β-Catenin, the investigators sought to determine if Rapgef5
affects one of these steps. To do so, they took advantage of
the fact that ectopic ventral expression of β-Catenin induces
a secondary axis in Xenopus embryos. Depleting rapgef5
in embryos in which wild-type β-Catenin was ectopically
expressed reduced the number of embryos with secondary
axes. Repeating this experiment with a mutated β-Catenin
such that it could not be phosphorylated by the β-Catenin
degradation complex and then degraded showed that
RAPGEF5 blocked the formation of secondary axes, suggesting that rapgef5 functions downstream of β-Catenin
degradation. Repeating this experiment with a β-Catenin
containing the Importin α/β1-dependent nuclear localization
signal prevented rapgef5 from blocking secondary axis formation. These experiments indicate that rapgef5 plays a role
in β-Catenin nuclear transport.
Based on these results, a model for a Rap-based
nuclear transport system was proposed. In support of
this model, endogenous Rapgef5 protein and active Raps
were found to localize to the nucleus, and Rap1 physically interacted with β-Catenin. Overexpressing constitutively active Rap1b rescued reduced foxj1 expression in
rapgef5-depleted gastrulae, whereas a dominant-negative
permanently GDP-bound Rap1b did not. Taken together,
these results support the existence and utilization of an
alternate energy-dependent nuclear transport pathway by
which β-Catenin can access the nucleus independently
of the classical Importin α/β1-mediated Ran-dependent
nuclear transport pathway (Griffn et al. 2018). Though
this began with a CHD/HTX candidate gene, the developmental study of rapgef5 opened the door to re-evaluating
the β-Catenin nuclear transport pathway, with implications for other signaling effectors.
16.4.4. SHARED MOLECULAR PATHWAYS FOR MULTIPLE
DISEASES: NEURODEVELOPMENTAL DISORDERS,
CRANIOFACIAL ABNORMALITIES, AND CHD
An important fnding from these studies inspired by patients
is that genes thought to have fundamental functions in cell
biology can have remarkably tissue-specifc effects in human
congenital malformations. For example, ribosomopathies
can lead to craniofacial malformations, a far cry from the
expectation that ribosomes are essential for protein production in all cells and therefore cell viability. Here we describe
the role of protein folding that affects many tissues but due
to a specifc effect on a remarkable population of cells called
the neural crest that affects cardiac development.
Variants in the gene encoding the endoplasmic reticulum
membrane protein complex subunit 1 (EMC1) were initially
identifed in a patient with retinitis pigmentosa (Abu-Saf eh
et al. 2013) and later expanded to include patients with neurodevelopmental defects, visual impairments, craniofacial
abnormalities, and CHD (Harel et al. 2016; Geetha et al.
2018; Homsy et al. 2015; Jin et al. 2017). The variants for
this broad cohort appear to be randomly dispersed across
the gene. Given that EMC1 is critical for the folding of multipass transmembrane proteins, how do we explain these phenotypes? This was frst investigated by depleting emc1 in X.
tropicalis and cataloging any developmental defects in the
resulting embryos. Defects in craniofacial cartilage development, cardiac outfow tract diameter, and embryo motility
were seen, recapitulating many of the patient phenotypes.
While wild-type human EMC1 mRNA rescued each of
these phenotypes in depleted embryos, mRNAs containing
patient variants did not, supporting the role of EMC1 defects
in each of these patient’s disease manifestations (Marquez
et al. 2020).
Craniofacial, neural, and cardiac progenitors are derived
from the neural crest cells (NCCs). Indeed, emc1 morphants
and embryos expressing disease variants had defects in NCC
gene expression and migration.
Label-free quantitative mass spectrometry (LFQMS) of
emc1 morphants compared to WT embryos revealed a loss
of proteins in the Wnt pathway, which is a critical pathway
for NCC specifcation (Dorsky, Moon, and Raible 1998; Maj
et al. 2016). As EMC1 plays a role in regulating multi-pass
membrane proteins, an obvious candidate was the Frizzled2
receptor (Fzd2). Immunofuorescence microscopy in RPE
cells after siRNA-mediated emc1 depletion revealed a loss
of membrane bound Fzd2 and Fzd7 and an increase in an
intracellular punctate signal characteristic of misfolded proteins. These results suggested that the NCC defects in emc1depleted embryos may be due to defects in proper folding
and membrane integration of Fzd receptors. In support of
this, when β-Catenin was expressed in NCCs, the loss of
neural crest specifcation was rescued.
In addition to the NCC-related defects, several patients
with EMC1 mutations also had visual impairments and
neurodevelopmental defects. Rhodopsin and the nicotinic acetylcholine receptor (nAChR) are also multi-pass
Discovering the Function of CHD Genes
but no role for it in embryonic development was known
( Griffn et al. 2018). Depleting rapgef5 in X. tropicalis
embryos led to abnormal cardiac looping similar to the
patient phenotype, suggesting a role in early development.
Indeed, rapgef5 depletion also affected earlier markers
of LR patterning, pitx2 and dand5. However, unlike the
two previous examples of CHD candidate genes, rapgef5
depletion reduced dand5 expression even before the commencement of cilia-driven fow. This suggested that rapgef5 depletion affected the specifcation of the LRO prior
to cilia-driven signaling.
The LRO is a transient mesodermal structure that is
overlaid by the endoderm as gastrulation is completed.
Therefore, any defects in gastrulation or formation of the
mesoderm can affect the formation of the LRO and LR signaling. As depletion of rapgef5 affected the formation of the
LRO, investigators examined other markers of the mesoderm and found that most were normal, except the known
Wnt responsive genes foxj1 and nodal3.1. These results indicated that rapgef5 likely disrupts LRO patterning and subsequent LR patterning by impacting Wnt signaling. Consistent
with this f nding, rapgef5 depletion reduced embryonic
β-Catenin levels. Given the many genes involved in transducing the Wnt signal into the cell and the degradation of
β-Catenin, the investigators sought to determine if Rapgef5
affects one of these steps. To do so, they took advantage of
the fact that ectopic ventral expression of β-Catenin induces
a secondary axis in Xenopus embryos. Depleting rapgef5
in embryos in which wild-type β-Catenin was ectopically
expressed reduced the number of embryos with secondary
axes. Repeating this experiment with a mutated β-Catenin
such that it could not be phosphorylated by the β-Catenin
degradation complex and then degraded showed that
RAPGEF5 blocked the formation of secondary axes, suggesting that rapgef5 functions downstream of β-Catenin
degradation. Repeating this experiment with a β-Catenin
containing the Importin α/β1-dependent nuclear localization
signal prevented rapgef5 from blocking secondary axis formation. These experiments indicate that rapgef5 plays a role
in β-Catenin nuclear transport.
Based on these results, a model for a Rap-based
nuclear transport system was proposed. In support of
this model, endogenous Rapgef5 protein and active Raps
were found to localize to the nucleus, and Rap1 physically interacted with β-Catenin. Overexpressing constitutively active Rap1b rescued reduced foxj1 expression in
rapgef5-depleted gastrulae, whereas a dominant-negative
permanently GDP-bound Rap1b did not. Taken together,
these results support the existence and utilization of an
alternate energy-dependent nuclear transport pathway by
which β-Catenin can access the nucleus independently
of the classical Importin α/β1-mediated Ran-dependent
nuclear transport pathway (Griffn et al. 2018). Though
this began with a CHD/HTX candidate gene, the developmental study of rapgef5 opened the door to re-evaluating
the β-Catenin nuclear transport pathway, with implications for other signaling effectors.
16.4.4. SHARED MOLECULAR PATHWAYS FOR MULTIPLE
DISEASES: NEURODEVELOPMENTAL DISORDERS,
CRANIOFACIAL ABNORMALITIES, AND CHD
An important fnding from these studies inspired by patients
is that genes thought to have fundamental functions in cell
biology can have remarkably tissue-specifc effects in human
congenital malformations. For example, ribosomopathies
can lead to craniofacial malformations, a far cry from the
expectation that ribosomes are essential for protein production in all cells and therefore cell viability. Here we describe
the role of protein folding that affects many tissues but due
to a specifc effect on a remarkable population of cells called
the neural crest that affects cardiac development.
Variants in the gene encoding the endoplasmic reticulum
membrane protein complex subunit 1 (EMC1) were initially
identifed in a patient with retinitis pigmentosa (Abu-Saf eh
et al. 2013) and later expanded to include patients with neurodevelopmental defects, visual impairments, craniofacial
abnormalities, and CHD (Harel et al. 2016; Geetha et al.
2018; Homsy et al. 2015; Jin et al. 2017). The variants for
this broad cohort appear to be randomly dispersed across
the gene. Given that EMC1 is critical for the folding of multipass transmembrane proteins, how do we explain these phenotypes? This was frst investigated by depleting emc1 in X.
tropicalis and cataloging any developmental defects in the
resulting embryos. Defects in craniofacial cartilage development, cardiac outfow tract diameter, and embryo motility
were seen, recapitulating many of the patient phenotypes.
While wild-type human EMC1 mRNA rescued each of
these phenotypes in depleted embryos, mRNAs containing
patient variants did not, supporting the role of EMC1 defects
in each of these patient’s disease manifestations (Marquez
et al. 2020).
Craniofacial, neural, and cardiac progenitors are derived
from the neural crest cells (NCCs). Indeed, emc1 morphants
and embryos expressing disease variants had defects in NCC
gene expression and migration.
Label-free quantitative mass spectrometry (LFQMS) of
emc1 morphants compared to WT embryos revealed a loss
of proteins in the Wnt pathway, which is a critical pathway
for NCC specifcation (Dorsky, Moon, and Raible 1998; Maj
et al. 2016). As EMC1 plays a role in regulating multi-pass
membrane proteins, an obvious candidate was the Frizzled2
receptor (Fzd2). Immunofuorescence microscopy in RPE
cells after siRNA-mediated emc1 depletion revealed a loss
of membrane bound Fzd2 and Fzd7 and an increase in an
intracellular punctate signal characteristic of misfolded proteins. These results suggested that the NCC defects in emc1depleted embryos may be due to defects in proper folding
and membrane integration of Fzd receptors. In support of
this, when β-Catenin was expressed in NCCs, the loss of
neural crest specifcation was rescued.
In addition to the NCC-related defects, several patients
with EMC1 mutations also had visual impairments and
neurodevelopmental defects. Rhodopsin and the nicotinic acetylcholine receptor (nAChR) are also multi-pass