181
Chromatin Remodeling during Development
There is not much detailed information available on
DNA methylation dynamics during the earliest stages of
Xenopus development. In zebrafsh, for example, the oocyte
and sperm patterns of DNA methylation differ. In cleavagestage fsh embryos, the maternal patterns are remodeled to
mimic those found in sperm and later stages of development (Jiang et al. 2013; Potok et al. 2013). Different from
pre-implantation development of mammals, neither zebrafish nor Xenopus exhibit a global genome-wide DNA hypomethylation around ZGA. Based on Southern blotting in
combination with methylation-sensitive restriction enzymes,
it was found that X. laevis genomic DNA is globally hypermethylated from early blastula (stage 6) onwards, similar to
sperm (Veenstra and Wolffe 2001). In mouse, Tet1, a dioxygenase enzyme involved in DNA demethylation, demethylates the genome after fertilization. Xenopus, however, do not
have an ortholog of mammalian Tet1. Xenopus species do
have Tet2 and Tet3, which are abundantly expressed from
neurula stages onwards. Based on morpholino injections,
Tet3 is important for eye and neural development (Xu et al.
2012). Whole genome bisulfte sequencing (WGBS) has
been performed from late blastula stages (stage 9) onward,
confrming the overall stability of DNA methylation over
developmental time (Hontelez et al. 2015; Bogdanovic et al.
2016). Around 600 enhancer elements, however, are actively
demethylated around stage 30, at the so-called phylotypic
stage. A similar phenomenon is observed in zebraf sh and
mouse embryos (Bogdanovic et al. 2016).
11.5.2. DNA METHYLATION, REPRESSION AND CROSSTALK WITH HISTONE MODIFICATIONS
DNA methylation is generally thought to be repressive
towards transcription. Using morpholinos in X. laevis
embryos to knock down the DNA methyl transferase Dnmt1,
the protein was found to contribute to repression of transcription before the mid-blastula transition. A catalytically
dead form of the protein, however, could rescue this phenotype, showing that this function is unrelated to DNA methylation (Dunican et al. 2008). Although DNA methylation
of CpG-dense promoters is strongly repressive in oocytes
(Jones et al. 1998), this is not necessarily true in blastulaand gastrula-stage embryos. In a comparison of methylated
versus unmethylated transgenes, methylated reporters were
strongly repressed in oocytes and tailbud-stage embryos
but not in blastula and gastrula embryos where they were
expressed despite being methylated (Bogdanovic et al. 2011).
This does not mean that DNA methylation is inconsequential in early development, as the H3K4me3 and H3K27me3
histone modifcations preferentially accumulate in hypomethylated regions from blastula stages onwards (Hontelez
et al. 2015).
11.6. FUTURE DIRECTIONS
Research in Xenopus has been at the frontier of chromatin research. As discussed previously, this included early
discoveries of the histone proteins as the packaging material
of genomic DNA, key insights in cloning and nuclear transfer experiments, and the frst epigenome maps of vertebrate
embryos. There are promising indications that Xenopus will
continue to play this role. Recently the chromosome topology
of chromosomal DNA during development has been described
(Quigley and Kintner 2017; Niu et al. 2021). Moreover, single
cell transcriptomics and single-cell chromatin accessibility
assays allow resolving gene regulatory landscape at the level
of individual cells, providing the resolution to disentangle
developmental trajectories and gene regulation in embryos at
the level of individual cell types (Briggs et al. 2018; Kakebeen
et al. 2020; Bright et al. 2021). New insights can also be gleaned
from comparisons between X. laevis and X. tropicalis. These
two species are generally considered similar in their developmental programs. Comparisons between the two species are
interesting in the context of genome evolution. X. laevis as
a species originated from inter-specifc hybridization 17–18
million years ago, resulting in a duplicate, allo-tetraploid
genome (Session et al. 2016). Kilobase-sized deletions are
prevalent in this genome, especially in chromosomes derived
from one of the parental species. In addition, reactivation of
transposons may have played a major role in reshaping the
epigenomic landscape (Elurbe et al. 2017). This indicates that
epigenomic comparisons of Xenopus species may contribute
new insights in genome evolution and chromatin dynamics
over ultra-long time scales.
All these exciting developments have been shaped in
some way or another by new technologies. Computational
analyses have become essential to extract insight from everincreasing amounts of data. All these developments and
compelling recent fndings on chromatin dynamics indicate
that Xenopus species have been and will continue to be powerful models for development and disease.
REFERENCES
Afouda BA, Nakamura Y, Shaw S, Charney RM, Paraiso KD, Blitz
IL, Cho KWY, Hoppler S. 2020. Foxh1/nodal def nes context-specifc direct maternal Wnt/β-catenin target gene regulation in early development. iScience 23 : 101314.
Akhtar W, Veenstra GJ. 2009. TBP2 is a substitute for TBP in
Xenopus oocyte transcription. BMC Biology 7 : 45.
Akkers RC, van Heeringen SJ, Jacobi UG, Janssen-Megens EM,
Francoijs KJ, Stunnenberg HG, Veenstra GJ. 2009. A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial
gene regulation in Xenopus embryos. Developmental Cell
17 : 425–434.
Alabert C, Barth TK, Reverón-Gómez N, Sidoli S, Schmidt A,
Jensen ON, Imhof A, Groth A. 2015. Two distinct modes for
propagation of histone PTMs across the cell cycle. Genes &
Development 29 : 585–590.
Amodeo AA, Jukam D, Straight AF, Skotheim JM. 2015, Mar
10. Histone titration against the genome sets the DNA-tocytoplasm threshold for the Xenopus midblastula transition. Proceedings of the National Academy of Sciences of
the United States of America112(10): E1086–E1095. doi:
10.1073/pnas.1413990112. Epub 2015 Feb 23. PMID:
25713373; PMCID: PMC4364222.
Chromatin Remodeling during Development
There is not much detailed information available on
DNA methylation dynamics during the earliest stages of
Xenopus development. In zebrafsh, for example, the oocyte
and sperm patterns of DNA methylation differ. In cleavagestage fsh embryos, the maternal patterns are remodeled to
mimic those found in sperm and later stages of development (Jiang et al. 2013; Potok et al. 2013). Different from
pre-implantation development of mammals, neither zebrafish nor Xenopus exhibit a global genome-wide DNA hypomethylation around ZGA. Based on Southern blotting in
combination with methylation-sensitive restriction enzymes,
it was found that X. laevis genomic DNA is globally hypermethylated from early blastula (stage 6) onwards, similar to
sperm (Veenstra and Wolffe 2001). In mouse, Tet1, a dioxygenase enzyme involved in DNA demethylation, demethylates the genome after fertilization. Xenopus, however, do not
have an ortholog of mammalian Tet1. Xenopus species do
have Tet2 and Tet3, which are abundantly expressed from
neurula stages onwards. Based on morpholino injections,
Tet3 is important for eye and neural development (Xu et al.
2012). Whole genome bisulfte sequencing (WGBS) has
been performed from late blastula stages (stage 9) onward,
confrming the overall stability of DNA methylation over
developmental time (Hontelez et al. 2015; Bogdanovic et al.
2016). Around 600 enhancer elements, however, are actively
demethylated around stage 30, at the so-called phylotypic
stage. A similar phenomenon is observed in zebraf sh and
mouse embryos (Bogdanovic et al. 2016).
11.5.2. DNA METHYLATION, REPRESSION AND CROSSTALK WITH HISTONE MODIFICATIONS
DNA methylation is generally thought to be repressive
towards transcription. Using morpholinos in X. laevis
embryos to knock down the DNA methyl transferase Dnmt1,
the protein was found to contribute to repression of transcription before the mid-blastula transition. A catalytically
dead form of the protein, however, could rescue this phenotype, showing that this function is unrelated to DNA methylation (Dunican et al. 2008). Although DNA methylation
of CpG-dense promoters is strongly repressive in oocytes
(Jones et al. 1998), this is not necessarily true in blastulaand gastrula-stage embryos. In a comparison of methylated
versus unmethylated transgenes, methylated reporters were
strongly repressed in oocytes and tailbud-stage embryos
but not in blastula and gastrula embryos where they were
expressed despite being methylated (Bogdanovic et al. 2011).
This does not mean that DNA methylation is inconsequential in early development, as the H3K4me3 and H3K27me3
histone modifcations preferentially accumulate in hypomethylated regions from blastula stages onwards (Hontelez
et al. 2015).
11.6. FUTURE DIRECTIONS
Research in Xenopus has been at the frontier of chromatin research. As discussed previously, this included early
discoveries of the histone proteins as the packaging material
of genomic DNA, key insights in cloning and nuclear transfer experiments, and the frst epigenome maps of vertebrate
embryos. There are promising indications that Xenopus will
continue to play this role. Recently the chromosome topology
of chromosomal DNA during development has been described
(Quigley and Kintner 2017; Niu et al. 2021). Moreover, single
cell transcriptomics and single-cell chromatin accessibility
assays allow resolving gene regulatory landscape at the level
of individual cells, providing the resolution to disentangle
developmental trajectories and gene regulation in embryos at
the level of individual cell types (Briggs et al. 2018; Kakebeen
et al. 2020; Bright et al. 2021). New insights can also be gleaned
from comparisons between X. laevis and X. tropicalis. These
two species are generally considered similar in their developmental programs. Comparisons between the two species are
interesting in the context of genome evolution. X. laevis as
a species originated from inter-specifc hybridization 17–18
million years ago, resulting in a duplicate, allo-tetraploid
genome (Session et al. 2016). Kilobase-sized deletions are
prevalent in this genome, especially in chromosomes derived
from one of the parental species. In addition, reactivation of
transposons may have played a major role in reshaping the
epigenomic landscape (Elurbe et al. 2017). This indicates that
epigenomic comparisons of Xenopus species may contribute
new insights in genome evolution and chromatin dynamics
over ultra-long time scales.
All these exciting developments have been shaped in
some way or another by new technologies. Computational
analyses have become essential to extract insight from everincreasing amounts of data. All these developments and
compelling recent fndings on chromatin dynamics indicate
that Xenopus species have been and will continue to be powerful models for development and disease.
REFERENCES
Afouda BA, Nakamura Y, Shaw S, Charney RM, Paraiso KD, Blitz
IL, Cho KWY, Hoppler S. 2020. Foxh1/nodal def nes context-specifc direct maternal Wnt/β-catenin target gene regulation in early development. iScience 23 : 101314.
Akhtar W, Veenstra GJ. 2009. TBP2 is a substitute for TBP in
Xenopus oocyte transcription. BMC Biology 7 : 45.
Akkers RC, van Heeringen SJ, Jacobi UG, Janssen-Megens EM,
Francoijs KJ, Stunnenberg HG, Veenstra GJ. 2009. A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial
gene regulation in Xenopus embryos. Developmental Cell
17 : 425–434.
Alabert C, Barth TK, Reverón-Gómez N, Sidoli S, Schmidt A,
Jensen ON, Imhof A, Groth A. 2015. Two distinct modes for
propagation of histone PTMs across the cell cycle. Genes &
Development 29 : 585–590.
Amodeo AA, Jukam D, Straight AF, Skotheim JM. 2015, Mar
10. Histone titration against the genome sets the DNA-tocytoplasm threshold for the Xenopus midblastula transition. Proceedings of the National Academy of Sciences of
the United States of America112(10): E1086–E1095. doi:
10.1073/pnas.1413990112. Epub 2015 Feb 23. PMID:
25713373; PMCID: PMC4364222.