319
Natural Genetic Variation and Disease
using the “isolated” clutch approach ( Figure 22.1C ). For
example, we used this approach to analyze the induction
of fetal growth restriction (FGR) resulting from ethanol
exposure or inhibition of RA biosynthesis ( Shukrun et
al., 2019 ). FGR is the Xenopus equivalent of intrauterine
growth restriction (IUGR), commonly observed in human
fetuses ( Cox and Marton, 2009 ; Gurugubelli Krishna and
Vishnu Bhat, 2018 ). IUGR can be subdivided into two
types: asymmetric, in which only the trunk displays growth
restriction, or symmetric, in which both the trunk and the
head are restricted ( Cox and Marton, 2009 ). Analysis of
multiple isolated clutches revealed that some exhibited
asymmetric and others symmetric FGR, recapitulating the
human disease in Xenopus embryos ( Shukrun et al., 2019 ).
IUGR, sometimes also called small for gestational age
(SGA), is defned as fetuses failing to reach their growth
potential during embryogenesis ( Mandruzzato et al., 2008 ;
Visser et al., 2014 ). Extreme IUGR cases can lead to disease and even lethality ( Bukowski, 2010 ; Gascoin and
Flamant, 2013 ; Mandruzzato et al., 2008 ). Similarly, analysis of the length of larvae generated from multiple isolated
clutches revealed a statistically signifcant dependence on
their maternal origin ( Figure 22.2B ), and the natural size
variability observed in Xenopus embryos paralleled the
size variability described for human fetuses ( Imdad et al.,
2011 ; Sharma et al., 2016 ). This use of the isolated clutch
approach is likely to continue to contribute to understanding the induction of IUGR and the genetic contribution
to this defect ( Sharma et al., 2017 ). This example demonstrates the usefulness of the outbred nature of Xenopus
frogs to explore the genetic impact in important human
health conditions.
22.3.2.3. Exploiting the Genetic Variability of
Xenopus for Disease Modeling
The Xenopus laevis genome provides an additional level
of genetic variation that can be exploited to further understand and model disease-causing genetic polymorphisms in
humans. Xenopus laevis arose around 17–18 million years
ago as an allotetraploid hybrid that carries distinct chromosomes derived from the two original species (Session et al.,
2016). Of the original gene pairs, one copy has been lost
in about 43% of protein-coding genes; the surviving singletons behave as in a diploid species. However, the remaining
protein-coding genes are present as homoeologue pairs with
different levels of subfunctionalization as a result of evolution (Hellsten et al., 2007; Session et al., 2016). While some
homoeologue pairs overlap extensively in expression pattern and level, sharing conserved protein product functions
(Kondo et al., 2017; Tour et al., 2001; Watanabe et al., 2017),
in other homoeologue pairs, these features have diverged,
often as the result of mutations in a regulatory element, similar to congenital-defect-causing mutations (Kondo et al.,
2017; Ochi et al., 2017a, 2017b; Watanabe et al., 2017). Can
we take advantage of the “enhanced” genetic diversif cation
of homoeologues to identify changes that resemble disease
conditions in humans?
Xenopus genetic variability can also provide insights into
the variability of human responses to environmental factors
and therapies. High-throughput chemical screens have been
performed to study the toxicity of environmental toxins and
drugs, as well as to identify small inhibitory molecules that
target specifc proteins or processes for functional studies
and drug discovery (Blay et al., 2020; Tomlinson et al., 2012).
Several chemical screens have been performed in Xenopus
to study signaling pathway components, angiogenesis, and
lymphangiogenesis (Kälin et al., 2009; Peterson et al.,
2006). Taking advantage of the natural genetic variation in
Xenopus laboratory colonies and the isolated clutch experimental design, variants exhibiting enhanced sensitivity or
resistance to a specifc small molecule can be identif ed.
These variants then can be compared to the polymorphisms
known in the homologous human protein to further inform
drug screening, development, and medical treatments.
The Xenopus oocyte also serves as an in vivo “test-tube”
to express proteins and analyze their function during normal
or disease conditions (see Chapter 9). The large size of the
oocyte and its effciency in translating exogenous, injected
mRNAs have made it an exemplary experimental system
in which to study secreted and membrane-bound proteins,
including channels and transporters (Marchant, 2018;
Mowry, 2020). Also, cellular processes, developmental
mechanisms, physiological events, molecular biology, viral
infection, and drug discovery have been studied in Xenopus
oocytes (Au et al., 2010; Lin-Moshier and Marchant, 2013;
Zeng et al., 2020). These studies have been expanded to
include proteins involved in abnormal physiological processes and neuropathological conditions (Baker et al., 2020;
Meyer et al., 2020; Nashimoto et al., 2020; Singh et al.,
2020). Importantly, injecting mRNA-encoding human proteins opens the possibility to directly study the effect of
genetic polymorphisms on protein function. Studies using
this approach have analyzed the effect of protein variants
on cholesterol transport, GABAp receptor function, and
Na/K pump function and on individuals suffering from
Bartter Syndrome type 3 (Baker et al., 2020; Meyer et al.,
2020; Nashimoto et al., 2020; Seys et al., 2017). Xenopus
oocytes also were utilized to transplant membranes from
Alzheimer’s patients to study their neuropathology and how
they differ (Singh et al., 2020).
RNAs encoding human protein variants can also be
injected into Xenopus embryos with the advantage that specifc tissues or organs can be targeted. For example, embryos
have been employed to study the role of RAD21 variants
in the induction of sclerocornea ( Zhang et al., 2019) and
the effects of SIX1 variants on craniofacial development
(Shah et al., 2020; Mehdizadeh et al., 2021). We recently
used this approach to identify polymorphisms in ALDH1a2
(RADLH2) that affect the level of enzymatic activity (Shabtai
et al., 2016). Analysis of embryos during late blastula, before
endogenous RA signaling begins, allows analysis of the
injected enzymatic activity with minimal endogenous background, whereas analysis during gastrula stages explores the
interaction of the human variants with the frog network
Natural Genetic Variation and Disease
using the “isolated” clutch approach ( Figure 22.1C ). For
example, we used this approach to analyze the induction
of fetal growth restriction (FGR) resulting from ethanol
exposure or inhibition of RA biosynthesis ( Shukrun et
al., 2019 ). FGR is the Xenopus equivalent of intrauterine
growth restriction (IUGR), commonly observed in human
fetuses ( Cox and Marton, 2009 ; Gurugubelli Krishna and
Vishnu Bhat, 2018 ). IUGR can be subdivided into two
types: asymmetric, in which only the trunk displays growth
restriction, or symmetric, in which both the trunk and the
head are restricted ( Cox and Marton, 2009 ). Analysis of
multiple isolated clutches revealed that some exhibited
asymmetric and others symmetric FGR, recapitulating the
human disease in Xenopus embryos ( Shukrun et al., 2019 ).
IUGR, sometimes also called small for gestational age
(SGA), is defned as fetuses failing to reach their growth
potential during embryogenesis ( Mandruzzato et al., 2008 ;
Visser et al., 2014 ). Extreme IUGR cases can lead to disease and even lethality ( Bukowski, 2010 ; Gascoin and
Flamant, 2013 ; Mandruzzato et al., 2008 ). Similarly, analysis of the length of larvae generated from multiple isolated
clutches revealed a statistically signifcant dependence on
their maternal origin ( Figure 22.2B ), and the natural size
variability observed in Xenopus embryos paralleled the
size variability described for human fetuses ( Imdad et al.,
2011 ; Sharma et al., 2016 ). This use of the isolated clutch
approach is likely to continue to contribute to understanding the induction of IUGR and the genetic contribution
to this defect ( Sharma et al., 2017 ). This example demonstrates the usefulness of the outbred nature of Xenopus
frogs to explore the genetic impact in important human
health conditions.
22.3.2.3. Exploiting the Genetic Variability of
Xenopus for Disease Modeling
The Xenopus laevis genome provides an additional level
of genetic variation that can be exploited to further understand and model disease-causing genetic polymorphisms in
humans. Xenopus laevis arose around 17–18 million years
ago as an allotetraploid hybrid that carries distinct chromosomes derived from the two original species (Session et al.,
2016). Of the original gene pairs, one copy has been lost
in about 43% of protein-coding genes; the surviving singletons behave as in a diploid species. However, the remaining
protein-coding genes are present as homoeologue pairs with
different levels of subfunctionalization as a result of evolution (Hellsten et al., 2007; Session et al., 2016). While some
homoeologue pairs overlap extensively in expression pattern and level, sharing conserved protein product functions
(Kondo et al., 2017; Tour et al., 2001; Watanabe et al., 2017),
in other homoeologue pairs, these features have diverged,
often as the result of mutations in a regulatory element, similar to congenital-defect-causing mutations (Kondo et al.,
2017; Ochi et al., 2017a, 2017b; Watanabe et al., 2017). Can
we take advantage of the “enhanced” genetic diversif cation
of homoeologues to identify changes that resemble disease
conditions in humans?
Xenopus genetic variability can also provide insights into
the variability of human responses to environmental factors
and therapies. High-throughput chemical screens have been
performed to study the toxicity of environmental toxins and
drugs, as well as to identify small inhibitory molecules that
target specifc proteins or processes for functional studies
and drug discovery (Blay et al., 2020; Tomlinson et al., 2012).
Several chemical screens have been performed in Xenopus
to study signaling pathway components, angiogenesis, and
lymphangiogenesis (Kälin et al., 2009; Peterson et al.,
2006). Taking advantage of the natural genetic variation in
Xenopus laboratory colonies and the isolated clutch experimental design, variants exhibiting enhanced sensitivity or
resistance to a specifc small molecule can be identif ed.
These variants then can be compared to the polymorphisms
known in the homologous human protein to further inform
drug screening, development, and medical treatments.
The Xenopus oocyte also serves as an in vivo “test-tube”
to express proteins and analyze their function during normal
or disease conditions (see Chapter 9). The large size of the
oocyte and its effciency in translating exogenous, injected
mRNAs have made it an exemplary experimental system
in which to study secreted and membrane-bound proteins,
including channels and transporters (Marchant, 2018;
Mowry, 2020). Also, cellular processes, developmental
mechanisms, physiological events, molecular biology, viral
infection, and drug discovery have been studied in Xenopus
oocytes (Au et al., 2010; Lin-Moshier and Marchant, 2013;
Zeng et al., 2020). These studies have been expanded to
include proteins involved in abnormal physiological processes and neuropathological conditions (Baker et al., 2020;
Meyer et al., 2020; Nashimoto et al., 2020; Singh et al.,
2020). Importantly, injecting mRNA-encoding human proteins opens the possibility to directly study the effect of
genetic polymorphisms on protein function. Studies using
this approach have analyzed the effect of protein variants
on cholesterol transport, GABAp receptor function, and
Na/K pump function and on individuals suffering from
Bartter Syndrome type 3 (Baker et al., 2020; Meyer et al.,
2020; Nashimoto et al., 2020; Seys et al., 2017). Xenopus
oocytes also were utilized to transplant membranes from
Alzheimer’s patients to study their neuropathology and how
they differ (Singh et al., 2020).
RNAs encoding human protein variants can also be
injected into Xenopus embryos with the advantage that specifc tissues or organs can be targeted. For example, embryos
have been employed to study the role of RAD21 variants
in the induction of sclerocornea ( Zhang et al., 2019) and
the effects of SIX1 variants on craniofacial development
(Shah et al., 2020; Mehdizadeh et al., 2021). We recently
used this approach to identify polymorphisms in ALDH1a2
(RADLH2) that affect the level of enzymatic activity (Shabtai
et al., 2016). Analysis of embryos during late blastula, before
endogenous RA signaling begins, allows analysis of the
injected enzymatic activity with minimal endogenous background, whereas analysis during gastrula stages explores the
interaction of the human variants with the frog network