210
Xenopus
2007). First, both Xenopus and murine pdx1 genes acted
similarly in converting liver to pancreas in the Xenopus
tadpole, but both proteins required an extra VP16 activation
domain. The ability of other pancreatic transcription factors
was tested similarly, and only one other transcription factor,
Ptf1a, had similar activity. These results reveal the power of
using multiple transgenes to test the functional ability of different factors in vivo in a tissue-specifc manner allowing for
analysis of cell fate conversions and commitment.
One of the major embryological benefts of Xenopus is
transplantation that creates chimeric embryos, and transgenic Xenopus embryos have proven very useful for these
experiments. Creating chimeric embryos between wild type
and transgenic embryos has been used to study pancreas
development, regeneration, and the origin of muscle satellite cells (Daughters et al., 2011; Gargioli and Slack, 2004;
Jarikji et al., 2009). The developing pancreas is derived
from separate dorsal and ventral buds arising from the roof
and foor of the archenteron. These buds can be selectively
labeled in chimeric embryos of wild type and pElas:GFP
transgenics. Using such an approach, Jarikji et al. (2009)
found that the ventral pancreatic cells migrate into the dorsal pancreas after fusion, whereas the dorsal pancreatic cells
do not. In another example, grafting specifc regions from
a transgenic CMV:GFP neurula stage embryo onto a wild
type host enabled Gargioli and Slack (2004) to follow the
fate of individual tissues (neural, notochord, or somites)
during tail regeneration. They found that notochord and spinal cord regenerate from the same tissue, whereas muscle
cells regenerate from a small population of satellite cells.
Other lines that are benefcial for such experiments include
ROSA26:GFP and Brainbow lines for long-term fate mapping (Gross et al., 2006).
In addition to following transplanted cells, transgenes can
be used to isolate specifc embryonic cells or nuclei, facilitating analysis of transcriptomes or proteomes. One method
uses two transgenes, one labeling nuclei of target cells with
a biotin ligase receptor and the second expressing the BirA
biotin ligase to biotinylate the target nuclei for isolation.
Using cell-type-specifc DNA elements, this approach generated proteomic profles of Xenopus cardiac nuclei (Amin
et al., 2014). Transgenic Xenopus in which particular cell
types are labeled were used to purify these cells for downstream analysis; this has relied on the speed and simplicity
of disaggregating cells in Xenopus prior to FACS sorting,
which was combined with RNAseq to identify key regulators of tail regeneration (Kakebeen et al., 2020).
Although these transgenic lines allow the biochemistry
of development to be studied, there are others that focus
more on cell biology. Some lines label various subcellular
structures, while others allow real-time spatiotemporal analysis of signaling pathway outputs, including Wnt, calcium,
epigenetic changes, and oxidative stress (Love et al., 2013;
Offner et al., 2020; Suzuki et al., 2016; Takagi et al., 2013;
Tran and Vleminckx, 2014).
Successful transgenic strategies depend on having identifed the appropriate DNA control elements to drive expression
of transgenes in the required temporal and spatial manner.
This has proven the most challenging element of transgene
design despite all of the information now available regarding
epigenetic modifcation and conservation of genomes (Kim
et al., 2019). While BAC and fosmid transgenesis have been
used successfully to address this challenge (Fish et al., 2011;
Ochi et al., 2012), gene editing, which is discussed in the
following, has produced an alternative approach that avoids
the challenges of handling large, fragile constructs and the
possibility of extremely distant control elements: inserting a
transgene into the endogenous locus so that its expression is
controlled by all of the DNA elements and epigenetic mechanisms that regulate the gene normally.
14.3. GENETICS AND GENE
EDITING IN XENOPUS
Traditionally, Xenopus were used mainly for embryological
experiments, with limited use as a genetic model. This was
largely due to the allotetraploidy of X. laevis, which was the
species used until recently; its long time to sexual maturity;
and the resulting amount of effort required to breed successive generations. The publication of the X. tropicalis genome
in 2009 and the X. laevis genome in 2016 together with new
genome editing technologies that have become available in
the last seven years have led to the generation of many new
Xenopus mutants. In this section, we outline a brief history
of Xenopus mutants and give an overview of gene editing
successes in Xenopus and future directions.
14.3.1. BEGINNINGS
Prior to the modern era of gene editing, naturally occurring
Xenopus mutants were identifed in the laboratory through
successive inbreeding or gynogenetic screens. Several
naturally occurring mutations were identifed and studied,
including anucleate, which lacked nucleoli and was instrumental in cloning ribosomal RNA genes (Elsdale et al., 1958;
Wallace, 1960). The second spontaneous X. laevis mutant
to be identifed was the periodic albinism mutant, which
produces white/yellow embryos that are excellent for gene
expression analysis and commonly used by the Xenopus
community (Hoperskaya, 1975). Recently, this mutation was
mapped to a 1.9kb deletion in the hps4 (Hermansky Pudlak
syndrome type 4) gene (Fukuzawa, 2021). Other developmental mutants were identifed in offspring derived from
nuclear transfer animals at the Geneva Xenopus Centre
(Droin, 1992). These naturally occurring mutants set the
foundation for future genetic studies in Xenopus.
14.3.2. X. TROPICALIS AND FORWARD GENETIC SCREENS
Interest in Xenopus genetics was reignited after the introduction to the laboratory of a closely related species, Xenopus
tropicalis (Abu-Daya et al., 2012). X. tropicalis is the only
diploid species in the genus, with one of the smallest known
haploid genomes, 1.5 × 10 9 bp in 10 chromosomes (2n = 20).
Xenopus
2007). First, both Xenopus and murine pdx1 genes acted
similarly in converting liver to pancreas in the Xenopus
tadpole, but both proteins required an extra VP16 activation
domain. The ability of other pancreatic transcription factors
was tested similarly, and only one other transcription factor,
Ptf1a, had similar activity. These results reveal the power of
using multiple transgenes to test the functional ability of different factors in vivo in a tissue-specifc manner allowing for
analysis of cell fate conversions and commitment.
One of the major embryological benefts of Xenopus is
transplantation that creates chimeric embryos, and transgenic Xenopus embryos have proven very useful for these
experiments. Creating chimeric embryos between wild type
and transgenic embryos has been used to study pancreas
development, regeneration, and the origin of muscle satellite cells (Daughters et al., 2011; Gargioli and Slack, 2004;
Jarikji et al., 2009). The developing pancreas is derived
from separate dorsal and ventral buds arising from the roof
and foor of the archenteron. These buds can be selectively
labeled in chimeric embryos of wild type and pElas:GFP
transgenics. Using such an approach, Jarikji et al. (2009)
found that the ventral pancreatic cells migrate into the dorsal pancreas after fusion, whereas the dorsal pancreatic cells
do not. In another example, grafting specifc regions from
a transgenic CMV:GFP neurula stage embryo onto a wild
type host enabled Gargioli and Slack (2004) to follow the
fate of individual tissues (neural, notochord, or somites)
during tail regeneration. They found that notochord and spinal cord regenerate from the same tissue, whereas muscle
cells regenerate from a small population of satellite cells.
Other lines that are benefcial for such experiments include
ROSA26:GFP and Brainbow lines for long-term fate mapping (Gross et al., 2006).
In addition to following transplanted cells, transgenes can
be used to isolate specifc embryonic cells or nuclei, facilitating analysis of transcriptomes or proteomes. One method
uses two transgenes, one labeling nuclei of target cells with
a biotin ligase receptor and the second expressing the BirA
biotin ligase to biotinylate the target nuclei for isolation.
Using cell-type-specifc DNA elements, this approach generated proteomic profles of Xenopus cardiac nuclei (Amin
et al., 2014). Transgenic Xenopus in which particular cell
types are labeled were used to purify these cells for downstream analysis; this has relied on the speed and simplicity
of disaggregating cells in Xenopus prior to FACS sorting,
which was combined with RNAseq to identify key regulators of tail regeneration (Kakebeen et al., 2020).
Although these transgenic lines allow the biochemistry
of development to be studied, there are others that focus
more on cell biology. Some lines label various subcellular
structures, while others allow real-time spatiotemporal analysis of signaling pathway outputs, including Wnt, calcium,
epigenetic changes, and oxidative stress (Love et al., 2013;
Offner et al., 2020; Suzuki et al., 2016; Takagi et al., 2013;
Tran and Vleminckx, 2014).
Successful transgenic strategies depend on having identifed the appropriate DNA control elements to drive expression
of transgenes in the required temporal and spatial manner.
This has proven the most challenging element of transgene
design despite all of the information now available regarding
epigenetic modifcation and conservation of genomes (Kim
et al., 2019). While BAC and fosmid transgenesis have been
used successfully to address this challenge (Fish et al., 2011;
Ochi et al., 2012), gene editing, which is discussed in the
following, has produced an alternative approach that avoids
the challenges of handling large, fragile constructs and the
possibility of extremely distant control elements: inserting a
transgene into the endogenous locus so that its expression is
controlled by all of the DNA elements and epigenetic mechanisms that regulate the gene normally.
14.3. GENETICS AND GENE
EDITING IN XENOPUS
Traditionally, Xenopus were used mainly for embryological
experiments, with limited use as a genetic model. This was
largely due to the allotetraploidy of X. laevis, which was the
species used until recently; its long time to sexual maturity;
and the resulting amount of effort required to breed successive generations. The publication of the X. tropicalis genome
in 2009 and the X. laevis genome in 2016 together with new
genome editing technologies that have become available in
the last seven years have led to the generation of many new
Xenopus mutants. In this section, we outline a brief history
of Xenopus mutants and give an overview of gene editing
successes in Xenopus and future directions.
14.3.1. BEGINNINGS
Prior to the modern era of gene editing, naturally occurring
Xenopus mutants were identifed in the laboratory through
successive inbreeding or gynogenetic screens. Several
naturally occurring mutations were identifed and studied,
including anucleate, which lacked nucleoli and was instrumental in cloning ribosomal RNA genes (Elsdale et al., 1958;
Wallace, 1960). The second spontaneous X. laevis mutant
to be identifed was the periodic albinism mutant, which
produces white/yellow embryos that are excellent for gene
expression analysis and commonly used by the Xenopus
community (Hoperskaya, 1975). Recently, this mutation was
mapped to a 1.9kb deletion in the hps4 (Hermansky Pudlak
syndrome type 4) gene (Fukuzawa, 2021). Other developmental mutants were identifed in offspring derived from
nuclear transfer animals at the Geneva Xenopus Centre
(Droin, 1992). These naturally occurring mutants set the
foundation for future genetic studies in Xenopus.
14.3.2. X. TROPICALIS AND FORWARD GENETIC SCREENS
Interest in Xenopus genetics was reignited after the introduction to the laboratory of a closely related species, Xenopus
tropicalis (Abu-Daya et al., 2012). X. tropicalis is the only
diploid species in the genus, with one of the smallest known
haploid genomes, 1.5 × 10 9 bp in 10 chromosomes (2n = 20).