236
Xenopus
recapitulated HTX phenotypes. All of these genes also
showed perturbations of LR patterning prior to cardiac
morphogenesis (Fakhro et al. 2011). This early analysis of
genetic contributions to HTX supported the need to cast a
wider net to identify a greater number of rare HTX and CHD
candidate genes to better understand CHD/HTX etiology.
As costs of next-generation whole genome and exome
sequencing have decreased dramatically over the past
decade, it has become more feasible than ever to use these
approaches for identifying gene candidates. The National
Heart, Lung and Blood Institute (NHLBI)’s Pediatric
Cardiac Genetic Consortium (PCGC) exploited whole
exome sequencing to do just that. The PCGC performed
de novo mutation analysis with WES in cases in which
patient-parent trios were available. Researchers looked for
high-probability single base pair substitutions and small
INDELs present in patient samples that were not present
in parent samples that are rare and deleterious (nonsense,
frameshift, splice-site, or damaging amino-acid altering
missense mutations). Additional single-nucleotide polymorphism (SNP) array analysis has helped identify rare CNVs
potentially contributing to patient CHD (Pediatric Cardiac
Genomics Consortium et al. 2013). Multiple alleles for candidate genes began to emerge over the duration of the study
from 2010 to 2015: 66 genes had two or more damaging de
novo mutations, and 19 had two or more loss-of-function
de novo mutations. Although this is a good starting point,
much work remains to identify a more comprehensive list
of CHD candidate genes. The most recent analysis of the
PCGC study found that larger cohort sizes (10,000 patients)
predict capture of 38% of CHD gene candidates (Jin et al.
2017). Therefore, patient cohort recruitment, genetic analysis, and mechanistic studies in model organisms will further
improve our understanding of CHD/HTX.
16.3. APPROACHES TO STUDYING
CHD/HTX IN XENOPUS
Following the identifcation of genes from CHD/HTX
patients, it is critical to identify plausible mechanisms that
lead to pathogenesis. Xenopus is a great model organism to
study CHD/HTX candidate genes in early development.
16.3.1. MORPHOLOGICAL AND DEVELOPMENTAL BENEFITS
OF STUDYING CHD/HTX IN XENOPUS
Xenopus are particularly useful for applying high-throughput genetic screening techniques for analyzing CHD/HTX
candidate genes for a variety of reasons. First, due to their
external development and transparency during organogenesis, researchers can easily view the developing heart and
internal organs without dissection. Additionally, with optical coherence tomography (OCT), rapid high resolution
internal anatomy of the heart is easily def ned (Deniz et al.
2017). The Xenopus heart is structurally similar to that of
humans: it has two atria separated by a septum, a single trabeculated ventricle, and an outfow tract. Because the fate
map of the early blastula embryos is well mapped, it is easy
to target manipulations to specifc subsets of cells via microinjection of mRNAs, antisense morpholino oligonucleotides
(MOs), or CRISPR/Cas9-mediated genome editing. For
example, an investigator can target one cell of the two-cell
embryo and subsequently select embryos in which the right
or the left side of the embryo is manipulated. This is unique
to Xenopus and is especially powerful for analyzing LR patterning (Blum et al. 2009).
16.3.2. USING CRISPR/CAS9 GENOME EDITING
AS A SCREENING TOOL IN XENOPUS
CRISPR/Cas9 genome editing is perhaps the most important
recent technology for using X. tropicalis as a tool for highthroughput screening. X. tropicalis has a diploid genome
( Offeld, Hirsch, and Grainger 2000), making it ideal for
CRISPR/Cas9 genome editing and establishment of mutant
lines. There are multiple tools for designing sgRNAs and
making sgRNAs effciently that work very well for Xenopus.
In addition, commercially available Cas9 protein makes
generating F0 knockouts quick, easy, and affordable, thus
enabling genetic screens. Once researchers identify candidate genes from patients, researchers can use CRISPR/Cas9
for preliminary screening of developmental defects and continue using it as a tool to probe the cell and molecular impacts
of the gene in development (Bhattacharya et al. 2015).
16.3.3. LEFT-RIGHT PATTERNING: A BEAUTIFUL PATHWAY TO
GUIDE CHD/HTX DISEASE MECHANISM ANALYSIS
The interrelation between CHD and HTX highlights the LR
patterning pathway as a powerful guide to begin analyzing
CHD/HTX gene candidates on a developmental timeline.
Briefy, LR patterning begins early in embryonic development at the left-right organizer (LRO), where beating of
motile cilia create a unidirectional leftward extracellular
f uid fow (Schweickert et al. 2010; Minegishi et al. 2017).
When the fow is sensed by immotile cilia at the periphery of
the LRO, the signal results in downregulation of the initially
symmetric Nodal antagonist, dand5, on the left side of the
LRO ( Blum et al. 2009). Repression of dand5 allows Nodal
signaling on the left side. This left-sided Nodal signaling at
the LRO is communicated to the left lateral plate mesoderm,
which results in left-sided expression of pitx2c (Blum et al.
2009 ; Schweickert et al. 2010 ; Desgrange, Le Garrec, and
Meilhac 2018). Lateral pitx2c expression then def nes asymmetric LR heart and organ orientation (Ryan et al. 1998).
Conveniently for study, each stage of the LR cascade has an
informative marker: cardiac looping, pitx2 expression, and
dand5 expression. Pinpointing the candidate gene’s role in a
characterized developmental pathway is particularly important for studying genes with no known function in early
development.
Several developmental signaling pathways are critical for
proper establishment of the LRO and for relaying signals later
in this LRO pathway. Canonical Wnt is necessary for LRO
Xenopus
recapitulated HTX phenotypes. All of these genes also
showed perturbations of LR patterning prior to cardiac
morphogenesis (Fakhro et al. 2011). This early analysis of
genetic contributions to HTX supported the need to cast a
wider net to identify a greater number of rare HTX and CHD
candidate genes to better understand CHD/HTX etiology.
As costs of next-generation whole genome and exome
sequencing have decreased dramatically over the past
decade, it has become more feasible than ever to use these
approaches for identifying gene candidates. The National
Heart, Lung and Blood Institute (NHLBI)’s Pediatric
Cardiac Genetic Consortium (PCGC) exploited whole
exome sequencing to do just that. The PCGC performed
de novo mutation analysis with WES in cases in which
patient-parent trios were available. Researchers looked for
high-probability single base pair substitutions and small
INDELs present in patient samples that were not present
in parent samples that are rare and deleterious (nonsense,
frameshift, splice-site, or damaging amino-acid altering
missense mutations). Additional single-nucleotide polymorphism (SNP) array analysis has helped identify rare CNVs
potentially contributing to patient CHD (Pediatric Cardiac
Genomics Consortium et al. 2013). Multiple alleles for candidate genes began to emerge over the duration of the study
from 2010 to 2015: 66 genes had two or more damaging de
novo mutations, and 19 had two or more loss-of-function
de novo mutations. Although this is a good starting point,
much work remains to identify a more comprehensive list
of CHD candidate genes. The most recent analysis of the
PCGC study found that larger cohort sizes (10,000 patients)
predict capture of 38% of CHD gene candidates (Jin et al.
2017). Therefore, patient cohort recruitment, genetic analysis, and mechanistic studies in model organisms will further
improve our understanding of CHD/HTX.
16.3. APPROACHES TO STUDYING
CHD/HTX IN XENOPUS
Following the identifcation of genes from CHD/HTX
patients, it is critical to identify plausible mechanisms that
lead to pathogenesis. Xenopus is a great model organism to
study CHD/HTX candidate genes in early development.
16.3.1. MORPHOLOGICAL AND DEVELOPMENTAL BENEFITS
OF STUDYING CHD/HTX IN XENOPUS
Xenopus are particularly useful for applying high-throughput genetic screening techniques for analyzing CHD/HTX
candidate genes for a variety of reasons. First, due to their
external development and transparency during organogenesis, researchers can easily view the developing heart and
internal organs without dissection. Additionally, with optical coherence tomography (OCT), rapid high resolution
internal anatomy of the heart is easily def ned (Deniz et al.
2017). The Xenopus heart is structurally similar to that of
humans: it has two atria separated by a septum, a single trabeculated ventricle, and an outfow tract. Because the fate
map of the early blastula embryos is well mapped, it is easy
to target manipulations to specifc subsets of cells via microinjection of mRNAs, antisense morpholino oligonucleotides
(MOs), or CRISPR/Cas9-mediated genome editing. For
example, an investigator can target one cell of the two-cell
embryo and subsequently select embryos in which the right
or the left side of the embryo is manipulated. This is unique
to Xenopus and is especially powerful for analyzing LR patterning (Blum et al. 2009).
16.3.2. USING CRISPR/CAS9 GENOME EDITING
AS A SCREENING TOOL IN XENOPUS
CRISPR/Cas9 genome editing is perhaps the most important
recent technology for using X. tropicalis as a tool for highthroughput screening. X. tropicalis has a diploid genome
( Offeld, Hirsch, and Grainger 2000), making it ideal for
CRISPR/Cas9 genome editing and establishment of mutant
lines. There are multiple tools for designing sgRNAs and
making sgRNAs effciently that work very well for Xenopus.
In addition, commercially available Cas9 protein makes
generating F0 knockouts quick, easy, and affordable, thus
enabling genetic screens. Once researchers identify candidate genes from patients, researchers can use CRISPR/Cas9
for preliminary screening of developmental defects and continue using it as a tool to probe the cell and molecular impacts
of the gene in development (Bhattacharya et al. 2015).
16.3.3. LEFT-RIGHT PATTERNING: A BEAUTIFUL PATHWAY TO
GUIDE CHD/HTX DISEASE MECHANISM ANALYSIS
The interrelation between CHD and HTX highlights the LR
patterning pathway as a powerful guide to begin analyzing
CHD/HTX gene candidates on a developmental timeline.
Briefy, LR patterning begins early in embryonic development at the left-right organizer (LRO), where beating of
motile cilia create a unidirectional leftward extracellular
f uid fow (Schweickert et al. 2010; Minegishi et al. 2017).
When the fow is sensed by immotile cilia at the periphery of
the LRO, the signal results in downregulation of the initially
symmetric Nodal antagonist, dand5, on the left side of the
LRO ( Blum et al. 2009). Repression of dand5 allows Nodal
signaling on the left side. This left-sided Nodal signaling at
the LRO is communicated to the left lateral plate mesoderm,
which results in left-sided expression of pitx2c (Blum et al.
2009 ; Schweickert et al. 2010 ; Desgrange, Le Garrec, and
Meilhac 2018). Lateral pitx2c expression then def nes asymmetric LR heart and organ orientation (Ryan et al. 1998).
Conveniently for study, each stage of the LR cascade has an
informative marker: cardiac looping, pitx2 expression, and
dand5 expression. Pinpointing the candidate gene’s role in a
characterized developmental pathway is particularly important for studying genes with no known function in early
development.
Several developmental signaling pathways are critical for
proper establishment of the LRO and for relaying signals later
in this LRO pathway. Canonical Wnt is necessary for LRO