26
Xenopus
FIGURE 3.1 The organization of the amphibian egg and embryonic patterning. (A) A frog egg at fertilization; the pigmented animal
pole is towards the top; the pale vegetal pole is towards the bottom.
(B) The egg at 20 minutes post-fertilization; the appearance of the
grey crescent is indicated by the arrow. (C) Model of cortical rotation (dorsal view of the egg); microtubules are shown as arrows. The
dotted line indicates the future midline (a, anterior; p, posterior). (D)
Spemann’s egg ligation experiments; the left panel shows a “strongly
constricted” egg at the early two cell stage; to the right are the two
main outcomes—isolation of the dorsal lip to one half (middle) or
bisection of the dorsal lip (right). (E) Twinned embryos result from
the right-hand case in D. (F) One normal embryo and one Bauchstück
(a ventralized “belly piece”) resulting from the middle case in D. (G)
Model of regional specifcation of the Xenopus blastula. Nieuwkoop
recombined zones I and II with zone IV to demonstrate mesoderm
induction in Xenopus (after Sudarwati and Nieuwkoop 1971).
Source: Panels (A–B) are reproduced from Rugh (1951); panels (D–F)
reproduced from Spemann (1924).
of this dorsal-ventral regionalization of the vegetal hemisphere in mesoderm and organizer induction (Ambystoma:
Boterenbrood and Nieuwkoop, 1973; Nieuwkoop, 1969;
Xenopus: Sudarwati and Nieuwkoop, 1971; Figure 3.1), and
(3) the presence of “germ plasm” in the vegetal cortex of
the frog egg (cytoplasme germinale; Bounoure, 1934 , 1931 ,
related to insect “pole plasm” (polares Plasma, Kahle, 1908 )
in germline specifcation (see Section 5).
This chapter reviews past work on the patterning of the
Xenopus embryo by maternal gene products, including the
discovery of localized RNAs in Xenopus oocytes (where
much of the initial progress was made); the mechanisms
of localization of these RNAs; and functional studies on
localized and non-localized molecules in germ layer specifcation, axis induction, and germline formation. These
ideas and their impact on their respective felds have been
reviewed separately or more comprehensively in the context
of overall vertebrate development and oocyte polarity, so I
will not present another broad comparative review. Rather,
my goal is to provide a general background to the main biological questions relating to the maternal control of Xenopus
development. I will also review the principal approaches and
fndings, following early work to current state of the art. The
sections can be read independently in any order; any omissions of material and references are unintentional and ref ect
limited space and my own view of the f eld.
3.2. MOLECULAR CHARACTERIZATION OF
MATERNAL AND LOCALIZED RNAS
Early discoveries in molecular biology identifed the central role of RNA in interpreting information encoded in the
DNA, with the defnitive discovery of mRNA being reported
in 1961 (Brenner et al., 1961; Gros et al., 1961; Hayashi
and Spiegelman, 1961; Jacob and Monod, 1961). The critical nature of maternal mRNA in development was inferred
through experiments in sea urchins and in frogs that showed
the importance of new protein synthesis rather than new
transcription in driving early development (Hultin, 1961;
Smith and Ecker, 1965; Tyler, 1965). More direct evidence
for maternal mRNA was obtained by cell-free translation
assays, showing that a minor proportion of Xenopus oocyte
total RNA could drive protein synthesis in cell-free translation assays (Davidson et al. 1966).
3.2.1. LOCALIZED MATERNAL MRNAS
Despite these demonstrations, the compelling notion that
specifc mRNAs might become specifcally localized and
direct the specifcation of different cell lineages remained
speculative (Davidson and Britten, 1971; Kalthoff, 1979).
However, the discovery that mRNAs are covalently modif ed
at the three-prime end with “polyadenylic acids” was both a
milestone in understanding gene expression regulation and
an advance in the detection and isolation of mRNAs (Darnell
et al., 1971; Edmonds et al., 1971; Lee et al., 1971). Analytical
hybridization of poly(A)+ RNAs with labeled cDNAs and
poly(U) in situ hybridization identifed asymmetric accumulation of (putative) mRNA in eggs of many species, including
Xenopus (Capco and Jeffery, 1981, 1979; Jeffery and Capco,
1978). Additional analysis of Xenopus vegetally enriched
cDNAs indicated that a minority of poly(A) RNA sequences
(about 3–5%) were enriched up to 20-fold at the vegetal pole
(Carpenter and Klein, 1982), likely representing localized
mRNAs. Importantly, cell-free translation of Xenopus oocyte
vegetal pole mRNAs identifed unique patterns of protein
synthesis (King and Barklis, 1985), implying the presence
of maternally localized transcripts with the ability to create
functional protein asymmetry in the embryo.
Advances in molecular cloning technology made it
possible to isolate and identify individual localized mRNA
sequences. Differential screening of an oocyte cDNA library
identifed one vegetally localized clone ( Rebagliati et al.,
1985 ), designated Vg1 (now gdf1). This molecule encodes a
member of the Transforming growth factor β (Tgfβ ) family
Xenopus
FIGURE 3.1 The organization of the amphibian egg and embryonic patterning. (A) A frog egg at fertilization; the pigmented animal
pole is towards the top; the pale vegetal pole is towards the bottom.
(B) The egg at 20 minutes post-fertilization; the appearance of the
grey crescent is indicated by the arrow. (C) Model of cortical rotation (dorsal view of the egg); microtubules are shown as arrows. The
dotted line indicates the future midline (a, anterior; p, posterior). (D)
Spemann’s egg ligation experiments; the left panel shows a “strongly
constricted” egg at the early two cell stage; to the right are the two
main outcomes—isolation of the dorsal lip to one half (middle) or
bisection of the dorsal lip (right). (E) Twinned embryos result from
the right-hand case in D. (F) One normal embryo and one Bauchstück
(a ventralized “belly piece”) resulting from the middle case in D. (G)
Model of regional specifcation of the Xenopus blastula. Nieuwkoop
recombined zones I and II with zone IV to demonstrate mesoderm
induction in Xenopus (after Sudarwati and Nieuwkoop 1971).
Source: Panels (A–B) are reproduced from Rugh (1951); panels (D–F)
reproduced from Spemann (1924).
of this dorsal-ventral regionalization of the vegetal hemisphere in mesoderm and organizer induction (Ambystoma:
Boterenbrood and Nieuwkoop, 1973; Nieuwkoop, 1969;
Xenopus: Sudarwati and Nieuwkoop, 1971; Figure 3.1), and
(3) the presence of “germ plasm” in the vegetal cortex of
the frog egg (cytoplasme germinale; Bounoure, 1934 , 1931 ,
related to insect “pole plasm” (polares Plasma, Kahle, 1908 )
in germline specifcation (see Section 5).
This chapter reviews past work on the patterning of the
Xenopus embryo by maternal gene products, including the
discovery of localized RNAs in Xenopus oocytes (where
much of the initial progress was made); the mechanisms
of localization of these RNAs; and functional studies on
localized and non-localized molecules in germ layer specifcation, axis induction, and germline formation. These
ideas and their impact on their respective felds have been
reviewed separately or more comprehensively in the context
of overall vertebrate development and oocyte polarity, so I
will not present another broad comparative review. Rather,
my goal is to provide a general background to the main biological questions relating to the maternal control of Xenopus
development. I will also review the principal approaches and
fndings, following early work to current state of the art. The
sections can be read independently in any order; any omissions of material and references are unintentional and ref ect
limited space and my own view of the f eld.
3.2. MOLECULAR CHARACTERIZATION OF
MATERNAL AND LOCALIZED RNAS
Early discoveries in molecular biology identifed the central role of RNA in interpreting information encoded in the
DNA, with the defnitive discovery of mRNA being reported
in 1961 (Brenner et al., 1961; Gros et al., 1961; Hayashi
and Spiegelman, 1961; Jacob and Monod, 1961). The critical nature of maternal mRNA in development was inferred
through experiments in sea urchins and in frogs that showed
the importance of new protein synthesis rather than new
transcription in driving early development (Hultin, 1961;
Smith and Ecker, 1965; Tyler, 1965). More direct evidence
for maternal mRNA was obtained by cell-free translation
assays, showing that a minor proportion of Xenopus oocyte
total RNA could drive protein synthesis in cell-free translation assays (Davidson et al. 1966).
3.2.1. LOCALIZED MATERNAL MRNAS
Despite these demonstrations, the compelling notion that
specifc mRNAs might become specifcally localized and
direct the specifcation of different cell lineages remained
speculative (Davidson and Britten, 1971; Kalthoff, 1979).
However, the discovery that mRNAs are covalently modif ed
at the three-prime end with “polyadenylic acids” was both a
milestone in understanding gene expression regulation and
an advance in the detection and isolation of mRNAs (Darnell
et al., 1971; Edmonds et al., 1971; Lee et al., 1971). Analytical
hybridization of poly(A)+ RNAs with labeled cDNAs and
poly(U) in situ hybridization identifed asymmetric accumulation of (putative) mRNA in eggs of many species, including
Xenopus (Capco and Jeffery, 1981, 1979; Jeffery and Capco,
1978). Additional analysis of Xenopus vegetally enriched
cDNAs indicated that a minority of poly(A) RNA sequences
(about 3–5%) were enriched up to 20-fold at the vegetal pole
(Carpenter and Klein, 1982), likely representing localized
mRNAs. Importantly, cell-free translation of Xenopus oocyte
vegetal pole mRNAs identifed unique patterns of protein
synthesis (King and Barklis, 1985), implying the presence
of maternally localized transcripts with the ability to create
functional protein asymmetry in the embryo.
Advances in molecular cloning technology made it
possible to isolate and identify individual localized mRNA
sequences. Differential screening of an oocyte cDNA library
identifed one vegetally localized clone ( Rebagliati et al.,
1985 ), designated Vg1 (now gdf1). This molecule encodes a
member of the Transforming growth factor β (Tgfβ ) family