29
Maternal mRNAs and Cell Lineages
the generation of viable homozygous mutant females or of
germline mosaics, a challenging prospect in amphibians.
Xenopus nevertheless offers several advantages relative
to other vertebrates for maternal gene analysis, including
(1) the ability to readily culture and manipulate oocytes
in vitro, (2) the effectiveness of DNA-based antisensemediated mRNA degradation in oocytes, and (3) the use
of oocyte/egg transfer procedures to ultimately fertilize
these oocytes. In addition, large-scale new zygotic mRNA
synthesis does not occur until the mid-blastula transition
(MBT; 4000-cell stage/stage 8), alongside other changes
in cell behavior (Newport and Kirschner, 1982), representing the main maternal-to-zygotic transition in Xenopus
(MZT; Vastenhouw et al., 2019). Thus, depleted maternal
mRNAs are unlikely to be replaced prior to early cell-fate
decisions being made. Together, these properties enabled
the development of powerful methods to use Xenopus to
examine vertebrate maternal gene functions (“oocyte hosttransfer”; Heasman et al., 1991; Houston, 2019, 2018; Mir and
Heasman, 2008; intracytoplasmic sperm injection: Miyamoto
et al., 2013, 2015a).
By the early 1970s, use of Xenopus oocytes became widespread owing to their use in the expression of heterologous
mRNAs (Gurdon et al., 1971). Previous work on oviductal
transport and species-specifc fertilization of amphibian
eggs also established methods for the transfer of coelomic
eggs between ovulating females (Rugh, 1935). These transplantation methods were later adapted to work with cultured
oocytes that were stimulated to mature in vitro as a test of
their developmental potential (Lithobates [née Rana] pipiens: Smith et al., 1968; Xenopus: Brun, 1975). Vegetal irradiation of cultured oocytes prior to host-transfer was used
to show the presence of UV-sensitive molecules important
for axis and germline formation (Elinson and Pasceri, 1989;
Holwill et al., 1987), establishing a paradigm for the prefertilization manipulation of development.
Xenopus oocytes were also an important test system for
antisense DNA oligonucleotide (oligo) technology in the
1980s. Oligonucleotides injected into full-grown oocytes
hybridize to complementary mRNAs and cleave the RNA
strand via endogenous RNase H (Cazenave et al., 1987; Dash
et al., 1987; Shuttleworth and Colman, 1988). However, the
same oligo types, even when modifed, were more toxic and
short lived when injected into fertilized eggs (Shuttleworth
et al., 1988; Woolf et al., 1990). This toxicity, along with
inherent limitations in the specifcity of RNase H-dependent
antisense DNA oligos (Woolf et al., 1992), discouraged the
widespread use of antisense technology in post-fertilization
Xenopus embryos until the commercial availability of
phosphorodiamidate morpholino oligos (“Morpholinos”;
Heasman et al., 2000; Summerton and Weller, 1997 ).
Janet Heasman and Chris Wylie frst coupled antisense
mRNA depletion in oocytes with fertilization through the host
transfer procedure (Heasman et al., 1991). They inaugurated
this method by depleting maternal cytokeratin (krt8.1 ) mRNA,
demonstrating that this gene was required for gastrulation
and wound healing in early embryos (Torpey et al., 1992).
Subsequent studies revealed a requirement for β-Catenin in
dorsal axis formation (Heasman et al., 1994). Because parallel experiments in Drosophila demonstrated that stabilization
of Armadillo/β-Catenin was a main output of Wnt signaling
(Peifer et al., 1994), these Xenopus antisense mRNA depletion
experiments strongly implicated endogenous maternal Wnt/
β-catenin activation in axis formation (Section 4).
This maternal mRNA depletion approach has been benefcial for understanding many aspects of early Xenopus
development because, in almost all cases, Morpholino
injection after fertilization at best only partially inhibits
maternally regulated processes and cannot affect processes
initiated around the time of injection. Another beneft of the
host-transfer method is that it can also be used to over- or
ectopically express proteins before fertilization, including
those representing genome editing reagents for F0 mutagenesis in Xenopus embryos (Aslan et al., 2017; Miyamoto et al.,
2015b; Nakajima and Yaoita, 2015; Ratzan et al., 2017).
3.4. MATERNAL CONTROL OF GERM LAYER
INDUCTION AND PATTERNING
Extensive work in Xenopus and other organisms has culminated in a largely unifed model for germ layer formation in
vertebrates (reviewed in Houston, 2017). This basic model
suggests that spatiotemporal gradients of Nodal signaling
(patterned by auto-regulation and by Wnt signals) induce
dorsal mesendoderm/organizer at high/early doses and ventrolateral mesendoderm at low/later doses. Organizer induction by Nodal occurs in synergy with early Wnt signaling
(maternal in Xenopus) and sets up self-regulating gradients
of BMP and later Wnt activity (zygotic in Xenopus) to pattern the dorsoventral and anteroposterior axes.
3.4.1. MATERNAL CONTROL OF ENDODERM
AND MESODERM BY VEGT
Nieuwkoop initially proposed that mesoderm (germ layer)
induction would involve new mRNA synthesis (Nieuwkoop,
1969), although some early experiments on heterochronic
blastomere recombinations and transplantations ( Dale and
Slack, 1987; Jones and Woodland, 1987) suggested a maternal
mesoderm inducer. The zygotic nature of germ layer induction was demonstrated by Wylie and Heasman, who showed
that vegetal masses only induced mesoderm after the onset
of zygotic transcription (Wylie et al., 1996 ). Several groups
subsequently and by different approaches identifed a likely
maternal transcription factor candidate, the T-domain transcription factor, Vegt, encoded by a vegetal cortex-localized mRNA (Horb and Thomsen, 1997; Lustig et al., 1996;
Stennard et al., 1996; Zhang and King, 1996 ).
The maternal role of vegt mRNA was assessed using
antisense oligos in host-transfer experiments ( Zhang et al.,
1998). These embryos largely lacked mesoderm and
endoderm, and vegetal cells gained expression of ectoderm
markers. Vegetal explants from vegt- depleted embryos
failed to induce mesoderm in animal caps (Zhang et al.,
Maternal mRNAs and Cell Lineages
the generation of viable homozygous mutant females or of
germline mosaics, a challenging prospect in amphibians.
Xenopus nevertheless offers several advantages relative
to other vertebrates for maternal gene analysis, including
(1) the ability to readily culture and manipulate oocytes
in vitro, (2) the effectiveness of DNA-based antisensemediated mRNA degradation in oocytes, and (3) the use
of oocyte/egg transfer procedures to ultimately fertilize
these oocytes. In addition, large-scale new zygotic mRNA
synthesis does not occur until the mid-blastula transition
(MBT; 4000-cell stage/stage 8), alongside other changes
in cell behavior (Newport and Kirschner, 1982), representing the main maternal-to-zygotic transition in Xenopus
(MZT; Vastenhouw et al., 2019). Thus, depleted maternal
mRNAs are unlikely to be replaced prior to early cell-fate
decisions being made. Together, these properties enabled
the development of powerful methods to use Xenopus to
examine vertebrate maternal gene functions (“oocyte hosttransfer”; Heasman et al., 1991; Houston, 2019, 2018; Mir and
Heasman, 2008; intracytoplasmic sperm injection: Miyamoto
et al., 2013, 2015a).
By the early 1970s, use of Xenopus oocytes became widespread owing to their use in the expression of heterologous
mRNAs (Gurdon et al., 1971). Previous work on oviductal
transport and species-specifc fertilization of amphibian
eggs also established methods for the transfer of coelomic
eggs between ovulating females (Rugh, 1935). These transplantation methods were later adapted to work with cultured
oocytes that were stimulated to mature in vitro as a test of
their developmental potential (Lithobates [née Rana] pipiens: Smith et al., 1968; Xenopus: Brun, 1975). Vegetal irradiation of cultured oocytes prior to host-transfer was used
to show the presence of UV-sensitive molecules important
for axis and germline formation (Elinson and Pasceri, 1989;
Holwill et al., 1987), establishing a paradigm for the prefertilization manipulation of development.
Xenopus oocytes were also an important test system for
antisense DNA oligonucleotide (oligo) technology in the
1980s. Oligonucleotides injected into full-grown oocytes
hybridize to complementary mRNAs and cleave the RNA
strand via endogenous RNase H (Cazenave et al., 1987; Dash
et al., 1987; Shuttleworth and Colman, 1988). However, the
same oligo types, even when modifed, were more toxic and
short lived when injected into fertilized eggs (Shuttleworth
et al., 1988; Woolf et al., 1990). This toxicity, along with
inherent limitations in the specifcity of RNase H-dependent
antisense DNA oligos (Woolf et al., 1992), discouraged the
widespread use of antisense technology in post-fertilization
Xenopus embryos until the commercial availability of
phosphorodiamidate morpholino oligos (“Morpholinos”;
Heasman et al., 2000; Summerton and Weller, 1997 ).
Janet Heasman and Chris Wylie frst coupled antisense
mRNA depletion in oocytes with fertilization through the host
transfer procedure (Heasman et al., 1991). They inaugurated
this method by depleting maternal cytokeratin (krt8.1 ) mRNA,
demonstrating that this gene was required for gastrulation
and wound healing in early embryos (Torpey et al., 1992).
Subsequent studies revealed a requirement for β-Catenin in
dorsal axis formation (Heasman et al., 1994). Because parallel experiments in Drosophila demonstrated that stabilization
of Armadillo/β-Catenin was a main output of Wnt signaling
(Peifer et al., 1994), these Xenopus antisense mRNA depletion
experiments strongly implicated endogenous maternal Wnt/
β-catenin activation in axis formation (Section 4).
This maternal mRNA depletion approach has been benefcial for understanding many aspects of early Xenopus
development because, in almost all cases, Morpholino
injection after fertilization at best only partially inhibits
maternally regulated processes and cannot affect processes
initiated around the time of injection. Another beneft of the
host-transfer method is that it can also be used to over- or
ectopically express proteins before fertilization, including
those representing genome editing reagents for F0 mutagenesis in Xenopus embryos (Aslan et al., 2017; Miyamoto et al.,
2015b; Nakajima and Yaoita, 2015; Ratzan et al., 2017).
3.4. MATERNAL CONTROL OF GERM LAYER
INDUCTION AND PATTERNING
Extensive work in Xenopus and other organisms has culminated in a largely unifed model for germ layer formation in
vertebrates (reviewed in Houston, 2017). This basic model
suggests that spatiotemporal gradients of Nodal signaling
(patterned by auto-regulation and by Wnt signals) induce
dorsal mesendoderm/organizer at high/early doses and ventrolateral mesendoderm at low/later doses. Organizer induction by Nodal occurs in synergy with early Wnt signaling
(maternal in Xenopus) and sets up self-regulating gradients
of BMP and later Wnt activity (zygotic in Xenopus) to pattern the dorsoventral and anteroposterior axes.
3.4.1. MATERNAL CONTROL OF ENDODERM
AND MESODERM BY VEGT
Nieuwkoop initially proposed that mesoderm (germ layer)
induction would involve new mRNA synthesis (Nieuwkoop,
1969), although some early experiments on heterochronic
blastomere recombinations and transplantations ( Dale and
Slack, 1987; Jones and Woodland, 1987) suggested a maternal
mesoderm inducer. The zygotic nature of germ layer induction was demonstrated by Wylie and Heasman, who showed
that vegetal masses only induced mesoderm after the onset
of zygotic transcription (Wylie et al., 1996 ). Several groups
subsequently and by different approaches identifed a likely
maternal transcription factor candidate, the T-domain transcription factor, Vegt, encoded by a vegetal cortex-localized mRNA (Horb and Thomsen, 1997; Lustig et al., 1996;
Stennard et al., 1996; Zhang and King, 1996 ).
The maternal role of vegt mRNA was assessed using
antisense oligos in host-transfer experiments ( Zhang et al.,
1998). These embryos largely lacked mesoderm and
endoderm, and vegetal cells gained expression of ectoderm
markers. Vegetal explants from vegt- depleted embryos
failed to induce mesoderm in animal caps (Zhang et al.,