33
Maternal mRNAs and Cell Lineages
germ cells (PGCs) could also be induced by signals acting on pluripotent cells, which may be the ancestral mode
(Extavour and Akam, 2003; Houston and King, 2000a).
Regardless of whether PGCs are specifed by maternal germ
plasm, a material similar to germ plasm has since been identifed in the differentiating adult germ cells of all animals
(typically termed nuage, Fr. “cloud”; André and Rouiller,
1956). Thus, germ plasm/nuage is likely a fundamental feature of the germline (Eddy, 1975, 1974). Recent experiments
on the reprogramming of somatic cells to PGCs have shown
that nuage does indeed form in reprogrammed cells (Bucay
et al., 2009), further suggesting that nuage is tightly connected to, and likely a product of, germ-cell differentiation.
Both descriptive and experimental studies in Rana/
Lithobates and in Xenopus eggs established that the vegetally
localized germ plasm is essential for germline formation in
Anuran amphibians (Houston and King, 2000a). The classical embryological evidence showed that UV-irradiation
of the vegetal pole severely reduced PGC formation (e.g.
Bounoure et al., 1954; Züst and Dixon, 1975). Unlike the case
in Drosophila (Ephrussi and Lehmann, 1992; Illmensee and
Mahowald, 1974), initial cytoplasmic transfer experiments
in Xenopus failed to show a similar strong determinative
role for germ plasm (Wakahara, 1977; Wylie et al., 1985).
Recent experiments, however, indicate that germ plasm
transplantation into animal pole cells can create functional
PGCs in genetically marked Xenopus (Tada et al., 2012),
although discrepancies with the previous studies remain
unresolved.
3.5.2. GERM PLASM MRNAS IN PGC SPECIFICATION
The frst germ-plasm mRNA to be studied functionally
in Xenopus was dazl, identif ed contemporaneously with
vegt (Houston et al., 1998). The Dazl family (Deleted in
AZoospermia-like) comprises a conserved group of RRM
domain RNA-binding proteins involved in many different
aspects of germ cell development in animals and in human
fertility (Fu et al., 2015; Kee et al., 2009). In Xenopus, dazl
exhibits a mitochondrial cloud-dependent mRNA localization pattern similar to nanos1 and remains detectable in
early PGCs through the tailbud stages (Houston et al., 1998;
Sekizaki et al., 2004). Maternal mRNA depletion studies
identifed a role for Dazl in PGC development in Xenopus,
with dazl-depleted embryos exhibiting PGC migration
defects and remaining abnormally clustered within the posterior endoderm (Houston and King, 2000b). Dazl is thus
thought to control competence for migration in PGCs either
directly or indirectly through an earlier step in establishing
PGC specifcation. Work in other systems has suggested
that Dazl likely functions in polyadenylation (Haston et al.,
2009; Smorag et al., 2014).
A number of additional genes localized to the germ plasm
have been identifed (Table 3.1). Several of these genes have
roles in PGC migration as assessed by Morpholino knockdown or antisense mRNA depletion. Notably, depletion
of mRNAs encoding RNA-binding proteins dnd1, ddx25,
nanos1, and dazl have similar effects (Horvay et al., 2006;
Houston and King, 2000b; Lai et al., 2012; Yamaguchi,
2013). The nature of these genes and mechanistic studies
of their effects on PGCs strongly suggest diverse roles in
RNA metabolism, with germ plasm components interacting
with and regulating each other to ultimately control PGC
fate and/or migration. As an example, Dnd1 has been implicated in the protection of target mRNAs (including itself)
from microRNAs (Kedde et al., 2007), in regulating Nanos1
translation (Aguero et al., 2018, 2017), and in the anchoring of localized trim36 mRNA to the vegetal cortex (Mei
et al., 2013). Recent experiments have also suggested that
additional pathways are involved in restricting the action
of Dnd1 (and possibly other germ plasm RNAs), including
the important role of the ubiquitin-independent proteasome
pathway, which is animally localized (Hwang et al., 2019).
Other germ plasm localized mRNAs, including sox7 and
efnb1, also have roles in PGC migration (Owens et al., 2017;
Butler et al., 2018).
In addition to controlling overall PGC specifcation, a number of mRNAs localized to the germ plasm appear to also act
in morphogenesis of the germ plasm itself. germes encodes a
Xenopus -specifc leucine zipper/EF-hand protein that interacts with dynein light chain (Berekelya et al., 2003, 2007)
and may control germ plasm morphology. Similarly, syntabulin (sybu), encoding a motor adaptor protein implicated in
mitochondrial transport in neurons and axis formation, is
involved in aggregation of germ plasm and in perinuclear
germ plasm relocalization in Xenopus, resulting in PGC
defciency (Colozza and Robertis, 2014; Oh and Houston,
2017b). Similarly, Grip2 is another vesicle transport-related
protein encoded by a germ plasm mRNA that is involved
in PGC development, although its role in germ plasm
morphogenesis has not been examined (Kaneshiro et al.,
2007; Kirilenko et al., 2008; Tarbashevich et al., 2007).
3.5.3. ASSEMBLY OF MATERNAL GERM PLASM
The assembly of germ plasm in vertebrates is not well understood. In zebraf sh, buckyball is necessary and suff cient for
germ plasm and mitochondrial cloud assembly (Bontems
et al., 2009). The homologous Xenopus gene, velo1, encodes
a vegetally localized mRNA that forms a major protein
component of the mitochondrial cloud in fsh and frog
(Boke et al., 2016; Claussen and Pieler, 2004; Heim et al.,
2014; Nijjar and Woodland, 2013a, 2013b). Recent data suggest that the structural role of Velo1 in the mitochondrial
cloud is linked to its ability to form amyloid fbrils via an
N-terminal prion-like domain (PLD; Boke et al., 2016).
Velo1 is also a highly disordered protein and contains a
domain at the C-terminus involved in non-specifc RNA binding. Disordered low-complexity protein domains are found
in numerous RNA-binding proteins, typically mediating the
formation of liquid hydrogel droplets (Kato and McKnight,
2016). Velo1 protein-protein interactions with Dazl and
Rbpms2 (and interactions among the cognate RNAs and
proteins) are also required for zebrafsh mitochondrial cloud
Maternal mRNAs and Cell Lineages
germ cells (PGCs) could also be induced by signals acting on pluripotent cells, which may be the ancestral mode
(Extavour and Akam, 2003; Houston and King, 2000a).
Regardless of whether PGCs are specifed by maternal germ
plasm, a material similar to germ plasm has since been identifed in the differentiating adult germ cells of all animals
(typically termed nuage, Fr. “cloud”; André and Rouiller,
1956). Thus, germ plasm/nuage is likely a fundamental feature of the germline (Eddy, 1975, 1974). Recent experiments
on the reprogramming of somatic cells to PGCs have shown
that nuage does indeed form in reprogrammed cells (Bucay
et al., 2009), further suggesting that nuage is tightly connected to, and likely a product of, germ-cell differentiation.
Both descriptive and experimental studies in Rana/
Lithobates and in Xenopus eggs established that the vegetally
localized germ plasm is essential for germline formation in
Anuran amphibians (Houston and King, 2000a). The classical embryological evidence showed that UV-irradiation
of the vegetal pole severely reduced PGC formation (e.g.
Bounoure et al., 1954; Züst and Dixon, 1975). Unlike the case
in Drosophila (Ephrussi and Lehmann, 1992; Illmensee and
Mahowald, 1974), initial cytoplasmic transfer experiments
in Xenopus failed to show a similar strong determinative
role for germ plasm (Wakahara, 1977; Wylie et al., 1985).
Recent experiments, however, indicate that germ plasm
transplantation into animal pole cells can create functional
PGCs in genetically marked Xenopus (Tada et al., 2012),
although discrepancies with the previous studies remain
unresolved.
3.5.2. GERM PLASM MRNAS IN PGC SPECIFICATION
The frst germ-plasm mRNA to be studied functionally
in Xenopus was dazl, identif ed contemporaneously with
vegt (Houston et al., 1998). The Dazl family (Deleted in
AZoospermia-like) comprises a conserved group of RRM
domain RNA-binding proteins involved in many different
aspects of germ cell development in animals and in human
fertility (Fu et al., 2015; Kee et al., 2009). In Xenopus, dazl
exhibits a mitochondrial cloud-dependent mRNA localization pattern similar to nanos1 and remains detectable in
early PGCs through the tailbud stages (Houston et al., 1998;
Sekizaki et al., 2004). Maternal mRNA depletion studies
identifed a role for Dazl in PGC development in Xenopus,
with dazl-depleted embryos exhibiting PGC migration
defects and remaining abnormally clustered within the posterior endoderm (Houston and King, 2000b). Dazl is thus
thought to control competence for migration in PGCs either
directly or indirectly through an earlier step in establishing
PGC specifcation. Work in other systems has suggested
that Dazl likely functions in polyadenylation (Haston et al.,
2009; Smorag et al., 2014).
A number of additional genes localized to the germ plasm
have been identifed (Table 3.1). Several of these genes have
roles in PGC migration as assessed by Morpholino knockdown or antisense mRNA depletion. Notably, depletion
of mRNAs encoding RNA-binding proteins dnd1, ddx25,
nanos1, and dazl have similar effects (Horvay et al., 2006;
Houston and King, 2000b; Lai et al., 2012; Yamaguchi,
2013). The nature of these genes and mechanistic studies
of their effects on PGCs strongly suggest diverse roles in
RNA metabolism, with germ plasm components interacting
with and regulating each other to ultimately control PGC
fate and/or migration. As an example, Dnd1 has been implicated in the protection of target mRNAs (including itself)
from microRNAs (Kedde et al., 2007), in regulating Nanos1
translation (Aguero et al., 2018, 2017), and in the anchoring of localized trim36 mRNA to the vegetal cortex (Mei
et al., 2013). Recent experiments have also suggested that
additional pathways are involved in restricting the action
of Dnd1 (and possibly other germ plasm RNAs), including
the important role of the ubiquitin-independent proteasome
pathway, which is animally localized (Hwang et al., 2019).
Other germ plasm localized mRNAs, including sox7 and
efnb1, also have roles in PGC migration (Owens et al., 2017;
Butler et al., 2018).
In addition to controlling overall PGC specifcation, a number of mRNAs localized to the germ plasm appear to also act
in morphogenesis of the germ plasm itself. germes encodes a
Xenopus -specifc leucine zipper/EF-hand protein that interacts with dynein light chain (Berekelya et al., 2003, 2007)
and may control germ plasm morphology. Similarly, syntabulin (sybu), encoding a motor adaptor protein implicated in
mitochondrial transport in neurons and axis formation, is
involved in aggregation of germ plasm and in perinuclear
germ plasm relocalization in Xenopus, resulting in PGC
defciency (Colozza and Robertis, 2014; Oh and Houston,
2017b). Similarly, Grip2 is another vesicle transport-related
protein encoded by a germ plasm mRNA that is involved
in PGC development, although its role in germ plasm
morphogenesis has not been examined (Kaneshiro et al.,
2007; Kirilenko et al., 2008; Tarbashevich et al., 2007).
3.5.3. ASSEMBLY OF MATERNAL GERM PLASM
The assembly of germ plasm in vertebrates is not well understood. In zebraf sh, buckyball is necessary and suff cient for
germ plasm and mitochondrial cloud assembly (Bontems
et al., 2009). The homologous Xenopus gene, velo1, encodes
a vegetally localized mRNA that forms a major protein
component of the mitochondrial cloud in fsh and frog
(Boke et al., 2016; Claussen and Pieler, 2004; Heim et al.,
2014; Nijjar and Woodland, 2013a, 2013b). Recent data suggest that the structural role of Velo1 in the mitochondrial
cloud is linked to its ability to form amyloid fbrils via an
N-terminal prion-like domain (PLD; Boke et al., 2016).
Velo1 is also a highly disordered protein and contains a
domain at the C-terminus involved in non-specifc RNA binding. Disordered low-complexity protein domains are found
in numerous RNA-binding proteins, typically mediating the
formation of liquid hydrogel droplets (Kato and McKnight,
2016). Velo1 protein-protein interactions with Dazl and
Rbpms2 (and interactions among the cognate RNAs and
proteins) are also required for zebrafsh mitochondrial cloud