226
Xenopus
nodal and its targets spreads rapidly in the left LPM and is
therefore called the Nodal cascade.
15.2.2. THE LEFT-RIGHT ORGANIZER
Left-sided Nodal cascade induction implies that left positional information was already generated by an earlier
mechanism, which is termed symmetry breakage. In
most vertebrates (fsh, frog, and mammals) but also in sea
urchins, cilia functions are ultimately linked to symmetry
breakage (Blum et al., 2009; Blum and Ott, 2018b; Little
and Norris, 2020). This notion is supported by the occurrence of heterotaxia/situs inversion in patients suffering
from ciliopathies. Later, it was postulated by Afzelius in
the 1980s that during development, motile cilia are required
for LR axis determination (Afzelius, 1981). An extensive
clinical picture of LR defects can be observed when specifcally cilia motility is affected, summarized as primary
ciliary dyskinesia (PCD; Wallmeier et al., 2020). Mutations
in the axonemal dynein 11, for example, are responsible for
Kartagener syndrome, which commonly includes the development of sinusitis, bronchiectasis, and laterality defects
(Afzelius, 1976). Based on a substantial body of work using
various model organisms and human genetics, a conserved
mechanism has been suggested for symmetry breakage. At
neurula stages, a small epithelium in the posterior midline
is characterized by the presence of motile mono-cilia and
was termed left-right organizer (LRO). LRO cilia rotate in
a clockwise fashion, which is unusual because most other
motile cilia show a stroke-type motion, for example, multiciliated cells of the respiratory tract. The molecular basis
of cilia rotation and determination of its direction is not
understood and remains to be elucidated. But, importantly, a
leftward f uid fow of extracellular fuids is generated, which
serves as positional information to specify the LR axis in
mammals, fsh, and amphibians but, interestingly, not in
reptiles and birds (Blum and Ott, 2018a ; Kajikawa et al.,
2020). In Xenopus, the gastrocoel roof plate (GRP; Shook
et al., 2004) constitutes the ciliated LRO, which develops
inside of neurula embryos in a dorso-posterior position of
the embryonic gut. Probably because of its hidden presence,
our identifcation of the frog LRO occurred almost 10 years
later than in mouse (Schweickert et al., 2007). The GRP
cells have a mesodermal fate and only transiently line the
gut before integrating into the notochord and somites (Shook
et al., 2004). Interestingly, this fate mirrors two distinct cell
populations which also differ in position and, importantly,
in function. Notochordal cells are found along the midline,
projecting motile and polarized cilia, whereas somitic cells
localize bilaterally and have non-polarized and non-motile
cilia ( Figure 15.1A; Boskovski et al., 2013 ; Schweickert et
al., 2007; Shook et al., 2004). This scenario is also found in
mouse (Little and Norris, 2020) and is interpreted as central
fow-generating and bilaterally sensory LRO cells (cLRO
and sLRO, respectively; Figure 15.1A).
The mechanism by which the directionality of leftward
fow is initially detected is still under debate. Two scenarios
have been proposed, which either favor a morphogen or a
mechano-sensory model. The frst concept implicates one or
more secreted factors being extracellularly transported to the
left, where they activate receptor-based signaling (Hirokawa
et al., 2012; Tanaka et al., 2005). The second model suggests that physical forces exerted by the fow result in a leftsided bending of non-motile, mechanosensory cilia, which
transduces a laterality def ning signal into the sensory cells
(McGrath et al., 2003; Shinohara and Hamada, 2017; Tabin
and Vogan, 2003). Although the picture of these upstream
processes is incomplete, the cellular and molecular target of
fow-induced signaling is well described.
15.2.3. THE LEFTWARD FLOW TARGET: DAND5
We identifed Dand5, a Cerberus-related, extracellular
Wnt, Bmp, and Nodal inhibitor, as the molecular component by which laterality is fxed (Schweickert et al., 2010).
Both dand5 and nodal are co-expressed in left and right
lateral sLRO cells (Vonica and Brivanlou, 2007). In postfow stages, dand5 mRNA is specif cally downregulated
in left sLRO cells. Reduction of dand5 mRNA is f ow
dependent because embryos with blocked cilia motility or
increased viscosity in the archenteron lose dand5 asymmetry (Schweickert et al., 2010). Hence, a model was proposed
in which Dand5 blocks the morphogen Nodal on both sides
in pre-fow stages. Due to fow-induced asymmetry, Dand5
concentrations decrease in left sLRO cells, and as a consequence, Nodal is released from suppression (Figure 15.1B;
Schweickert et al., 2010) and diffuses to the left LPM, resulting in Nodal cascade induction.
The frog system is particularly suited to analyze this
complex mechanism, as well as many other LR processes
(Blum et al., 2009). Besides many more general advantages
outlined in this book, one experimental feature unique to
the Xenopus embryo is the ability to specif cally target left
or right cell lineages via simple microinjections at early
blastomere stages (2–32 cells). Although this possibility is
of experimental value to study many developmental questions, its unique strength is particularly emphasized during
the analysis of the LR body axis (Blum et al., 2009; Blum
and Ott, 2019). Providing loss- and gain-of-function experiments in a one-sided manner allows one to decipher the signaling pathways and mechanisms required to establish LR
asymmetry. In addition, targeting along the dorso-ventral
axis allows one to manipulate tissue-specifc processes in
space and time. Figure 15.1C depicts a generalized injection
setup for targeting distinct LR-relevant tissues like the f owgenerating central cLRO cells (C1 lineage), the f ow-sensing
lateral sLRO cells of the C2 lineage, and the LPM cells (C3
lineage).
The relevance of such experimental options is highlighted
by our analysis of dand5 regulation. In this context, an antisense morpholino-oligo (MO) specifc against endogenous
dand5 mRNA (dand5 MO) turned out to be extremely valuable for analyzing fow-dependent processes. In the normal
scenario, dand5 is repressed in a f ow-dependent mechanism
Xenopus
nodal and its targets spreads rapidly in the left LPM and is
therefore called the Nodal cascade.
15.2.2. THE LEFT-RIGHT ORGANIZER
Left-sided Nodal cascade induction implies that left positional information was already generated by an earlier
mechanism, which is termed symmetry breakage. In
most vertebrates (fsh, frog, and mammals) but also in sea
urchins, cilia functions are ultimately linked to symmetry
breakage (Blum et al., 2009; Blum and Ott, 2018b; Little
and Norris, 2020). This notion is supported by the occurrence of heterotaxia/situs inversion in patients suffering
from ciliopathies. Later, it was postulated by Afzelius in
the 1980s that during development, motile cilia are required
for LR axis determination (Afzelius, 1981). An extensive
clinical picture of LR defects can be observed when specifcally cilia motility is affected, summarized as primary
ciliary dyskinesia (PCD; Wallmeier et al., 2020). Mutations
in the axonemal dynein 11, for example, are responsible for
Kartagener syndrome, which commonly includes the development of sinusitis, bronchiectasis, and laterality defects
(Afzelius, 1976). Based on a substantial body of work using
various model organisms and human genetics, a conserved
mechanism has been suggested for symmetry breakage. At
neurula stages, a small epithelium in the posterior midline
is characterized by the presence of motile mono-cilia and
was termed left-right organizer (LRO). LRO cilia rotate in
a clockwise fashion, which is unusual because most other
motile cilia show a stroke-type motion, for example, multiciliated cells of the respiratory tract. The molecular basis
of cilia rotation and determination of its direction is not
understood and remains to be elucidated. But, importantly, a
leftward f uid fow of extracellular fuids is generated, which
serves as positional information to specify the LR axis in
mammals, fsh, and amphibians but, interestingly, not in
reptiles and birds (Blum and Ott, 2018a ; Kajikawa et al.,
2020). In Xenopus, the gastrocoel roof plate (GRP; Shook
et al., 2004) constitutes the ciliated LRO, which develops
inside of neurula embryos in a dorso-posterior position of
the embryonic gut. Probably because of its hidden presence,
our identifcation of the frog LRO occurred almost 10 years
later than in mouse (Schweickert et al., 2007). The GRP
cells have a mesodermal fate and only transiently line the
gut before integrating into the notochord and somites (Shook
et al., 2004). Interestingly, this fate mirrors two distinct cell
populations which also differ in position and, importantly,
in function. Notochordal cells are found along the midline,
projecting motile and polarized cilia, whereas somitic cells
localize bilaterally and have non-polarized and non-motile
cilia ( Figure 15.1A; Boskovski et al., 2013 ; Schweickert et
al., 2007; Shook et al., 2004). This scenario is also found in
mouse (Little and Norris, 2020) and is interpreted as central
fow-generating and bilaterally sensory LRO cells (cLRO
and sLRO, respectively; Figure 15.1A).
The mechanism by which the directionality of leftward
fow is initially detected is still under debate. Two scenarios
have been proposed, which either favor a morphogen or a
mechano-sensory model. The frst concept implicates one or
more secreted factors being extracellularly transported to the
left, where they activate receptor-based signaling (Hirokawa
et al., 2012; Tanaka et al., 2005). The second model suggests that physical forces exerted by the fow result in a leftsided bending of non-motile, mechanosensory cilia, which
transduces a laterality def ning signal into the sensory cells
(McGrath et al., 2003; Shinohara and Hamada, 2017; Tabin
and Vogan, 2003). Although the picture of these upstream
processes is incomplete, the cellular and molecular target of
fow-induced signaling is well described.
15.2.3. THE LEFTWARD FLOW TARGET: DAND5
We identifed Dand5, a Cerberus-related, extracellular
Wnt, Bmp, and Nodal inhibitor, as the molecular component by which laterality is fxed (Schweickert et al., 2010).
Both dand5 and nodal are co-expressed in left and right
lateral sLRO cells (Vonica and Brivanlou, 2007). In postfow stages, dand5 mRNA is specif cally downregulated
in left sLRO cells. Reduction of dand5 mRNA is f ow
dependent because embryos with blocked cilia motility or
increased viscosity in the archenteron lose dand5 asymmetry (Schweickert et al., 2010). Hence, a model was proposed
in which Dand5 blocks the morphogen Nodal on both sides
in pre-fow stages. Due to fow-induced asymmetry, Dand5
concentrations decrease in left sLRO cells, and as a consequence, Nodal is released from suppression (Figure 15.1B;
Schweickert et al., 2010) and diffuses to the left LPM, resulting in Nodal cascade induction.
The frog system is particularly suited to analyze this
complex mechanism, as well as many other LR processes
(Blum et al., 2009). Besides many more general advantages
outlined in this book, one experimental feature unique to
the Xenopus embryo is the ability to specif cally target left
or right cell lineages via simple microinjections at early
blastomere stages (2–32 cells). Although this possibility is
of experimental value to study many developmental questions, its unique strength is particularly emphasized during
the analysis of the LR body axis (Blum et al., 2009; Blum
and Ott, 2019). Providing loss- and gain-of-function experiments in a one-sided manner allows one to decipher the signaling pathways and mechanisms required to establish LR
asymmetry. In addition, targeting along the dorso-ventral
axis allows one to manipulate tissue-specifc processes in
space and time. Figure 15.1C depicts a generalized injection
setup for targeting distinct LR-relevant tissues like the f owgenerating central cLRO cells (C1 lineage), the f ow-sensing
lateral sLRO cells of the C2 lineage, and the LPM cells (C3
lineage).
The relevance of such experimental options is highlighted
by our analysis of dand5 regulation. In this context, an antisense morpholino-oligo (MO) specifc against endogenous
dand5 mRNA (dand5 MO) turned out to be extremely valuable for analyzing fow-dependent processes. In the normal
scenario, dand5 is repressed in a f ow-dependent mechanism