228
Xenopus
in the left sLRO cells (Schweickert et al., 2010). Then, further diminishing the Dand5 activity by injecting dand5
MO into the left lineage does not impact LR asymmetry.
However, right-sided dand5 knockdown induces an ectopic
Nodal-cascade in the right LPM (Figure 15.1B). Both observations are in line with the key role of dand5 in assigning the
left positional information by its f ow-induced repression.
Importantly, basically all experimental manipulations of
upstream processes, that is, impairing fow or f ow-induced
signaling, are rescued by left-sided dand5 MO administrations (Figure 15.1B; Schweickert et al., 2010). Laterality
defects induced by preventing leftward fow and thus dand5
repression by increasing the viscosity of extracellular milieu
or by interfering with cilia motility are rescued to wild-type
levels by left-sided dand5 knockdown. When injected into
the right lineage, organ situs was inverted in almost 100%
of cases (Schweickert et al., 2010). Similarly, we could demonstrate that laterality defects in conjoined twins are based
on mispositioning of LRO tissue and thus misregulation of
dand5 (Tisler et al., 2017b). Therefore, using the dand5 MO
in combination with other treatments allows one to demonstrate the specifcity of the experiments, and, even more
importantly, it allows one to place a particular gene function
upstream or downstream of f ow-induced dand5 inhibition.
Any factor whose function is required for Dand5-mediated
repression of Nodal should be rescuable by Dand5 knockdown. So far, such fow effectors are missing. Unfortunately,
the best candidate available to date, pkd2, which encodes
a critical calcium channel of the ciliary sensor complex
(Yoshiba et al., 2012; Yoshiba and Hamada, 2014), is already
required for the induction of the LRO anlage at blastula/gastrula stages (Vick et al., 2009), preventing its analysis during
fow stages. Together with yet-to-be-identifed factors of the
fow-induced signaling pathway, a more detailed picture of
symmetry breakage is still missing.
15.2.4. POST-TRANSCRIPTIONAL REGULATION
OF THE DAND5 MRNA
As in Xenopus, fow-dependent Dand5 repression in mouse
and fsh embryos was experimentally demonstrated by a
left-sided reduction of dand5 mRNA (Hojo et al., 2007;
Nakamura et al., 2012). In mouse, it was shown that dand5
mRNA reduction was conveyed via its 3’UTR in a f ow
dependent manner (Nakamura et al., 2012), indicating a
post-transcriptional mechanism. However, timing and penetrance of dand5 asymmetry suggested that mRNA decay
might not be the sole regulatory mechanism that controls
Dand5 protein levels. This reasoning is backed by observations in frog embryos. At late neurula stages (st. 19–21)
dand5 asymmetry is most prevalently detected, although
the Nodal cascade was already active in the left LPM
(Schweickert et al., 2010). Thus, dand5 mRNA decay was
too late to be functionally relevant. Also, the frequency
of left-sided dand5 mRNA reduction is not complete and
can be detected in 80% of wild type specimens at most,
whereas leftness at the level of asymmetric gene expression and organogenesis was observed in 95% of cases.
Obviously, these frequencies do not match, supporting
the idea that fow-dependent and robust dand5 repression
requires an additional process beyond detectable left-sided
dand5 mRNA degradation. These observations prompted
our hypothesis that fow sensation triggers a dand5 mRNAspecifc post-transcriptional regulatory program, which acts
through translational repression followed by mRNA decay
(Maerker et al., 2020).
Because the mechanisms by which the fow is sensed
or by which the signaling pathway is initiated are unclear,
we decided to tackle the problem from another perspective and addressed the question of how dand5 is regulated
in a fow-dependent manner. As outlined previously, posttranscriptional inhibition of dand5 mRNA translation was a
very likely possibility, and we searched for factors that could
provide this activity. We identifed the RNA binding protein
Bicaudal C (Bicc1) as the crucial post-transcriptional regulator of dand5 (Maerker et al., 2020). To date, a variety of
Bicc1 functions have been reported, ranging from repression
to protection of mRNAs (Rothé et al., 2015; Tran et al., 2010;
Zhang et al., 2014, 2013). Interestingly, Bicc1 was already
implicated in LR asymmetry in mouse and frog because of
its requirement to polarize fow-generating LRO cilia via
planar cell polarity (Maisonneuve et al., 2009). However,
bicc1 is co-expressed with dand5 and nodal in sLRO cells
(Maisonneuve et al., 2009) and inhibits the translation of a
reporter mRNA containing the dand5 3’UTR ( Zhang et al.,
2013). We recently showed that 139 nucleotides of the most
proximal dand5 3’UTR were suffcient to mediate Bicc1
regulation; we termed the sequence the Bicc1 responsive
element (Bicc1RE; Maerker et al., 2020). In vivo, a target
protector MO (tpMO), which blocks accessibility to the
Bicc1RE, prevented left-sided Nodal cascade induction, suggesting that Bicc1 mediates dand5 repression. Remarkably,
f ow-dependent decay of dand5 mRNA was not affected by
the tpMO, strongly suggesting that translation inhibition is
central to Bicc1 function. We postulate that f ow-induced
signaling triggers a yet-unknown posttranslational modifcation of Bicc1 that switches its activity towards translational repression of dand5 (Figure 15.2; Maerker et al.,
2020). Interestingly, we demonstrated that Dicer, a ribonuclease that is critical for miR processing, was expressed
in sLRO cells and is required for f ow-induced dand5
repression. In agreement, dicer morphants lack asymmetric
Nodal cascade induction. We further showed that Bicc1 and
Dicer acted in a synergistic manner, suggesting that both
post-transcriptional mechanisms co-operate during symmetry breakage (Maerker et al., 2020). Taken together, our
Xenopus work provides a new framework for how symmetry breakage is implemented from cilia-generated leftward
f uid fow to the asymmetric release of the Nodal morphogen. Surprisingly, post-transcriptional regulation is the initial target of fow, which opens a new route for sophisticated
analysis in the future.
Xenopus
in the left sLRO cells (Schweickert et al., 2010). Then, further diminishing the Dand5 activity by injecting dand5
MO into the left lineage does not impact LR asymmetry.
However, right-sided dand5 knockdown induces an ectopic
Nodal-cascade in the right LPM (Figure 15.1B). Both observations are in line with the key role of dand5 in assigning the
left positional information by its f ow-induced repression.
Importantly, basically all experimental manipulations of
upstream processes, that is, impairing fow or f ow-induced
signaling, are rescued by left-sided dand5 MO administrations (Figure 15.1B; Schweickert et al., 2010). Laterality
defects induced by preventing leftward fow and thus dand5
repression by increasing the viscosity of extracellular milieu
or by interfering with cilia motility are rescued to wild-type
levels by left-sided dand5 knockdown. When injected into
the right lineage, organ situs was inverted in almost 100%
of cases (Schweickert et al., 2010). Similarly, we could demonstrate that laterality defects in conjoined twins are based
on mispositioning of LRO tissue and thus misregulation of
dand5 (Tisler et al., 2017b). Therefore, using the dand5 MO
in combination with other treatments allows one to demonstrate the specifcity of the experiments, and, even more
importantly, it allows one to place a particular gene function
upstream or downstream of f ow-induced dand5 inhibition.
Any factor whose function is required for Dand5-mediated
repression of Nodal should be rescuable by Dand5 knockdown. So far, such fow effectors are missing. Unfortunately,
the best candidate available to date, pkd2, which encodes
a critical calcium channel of the ciliary sensor complex
(Yoshiba et al., 2012; Yoshiba and Hamada, 2014), is already
required for the induction of the LRO anlage at blastula/gastrula stages (Vick et al., 2009), preventing its analysis during
fow stages. Together with yet-to-be-identifed factors of the
fow-induced signaling pathway, a more detailed picture of
symmetry breakage is still missing.
15.2.4. POST-TRANSCRIPTIONAL REGULATION
OF THE DAND5 MRNA
As in Xenopus, fow-dependent Dand5 repression in mouse
and fsh embryos was experimentally demonstrated by a
left-sided reduction of dand5 mRNA (Hojo et al., 2007;
Nakamura et al., 2012). In mouse, it was shown that dand5
mRNA reduction was conveyed via its 3’UTR in a f ow
dependent manner (Nakamura et al., 2012), indicating a
post-transcriptional mechanism. However, timing and penetrance of dand5 asymmetry suggested that mRNA decay
might not be the sole regulatory mechanism that controls
Dand5 protein levels. This reasoning is backed by observations in frog embryos. At late neurula stages (st. 19–21)
dand5 asymmetry is most prevalently detected, although
the Nodal cascade was already active in the left LPM
(Schweickert et al., 2010). Thus, dand5 mRNA decay was
too late to be functionally relevant. Also, the frequency
of left-sided dand5 mRNA reduction is not complete and
can be detected in 80% of wild type specimens at most,
whereas leftness at the level of asymmetric gene expression and organogenesis was observed in 95% of cases.
Obviously, these frequencies do not match, supporting
the idea that fow-dependent and robust dand5 repression
requires an additional process beyond detectable left-sided
dand5 mRNA degradation. These observations prompted
our hypothesis that fow sensation triggers a dand5 mRNAspecifc post-transcriptional regulatory program, which acts
through translational repression followed by mRNA decay
(Maerker et al., 2020).
Because the mechanisms by which the fow is sensed
or by which the signaling pathway is initiated are unclear,
we decided to tackle the problem from another perspective and addressed the question of how dand5 is regulated
in a fow-dependent manner. As outlined previously, posttranscriptional inhibition of dand5 mRNA translation was a
very likely possibility, and we searched for factors that could
provide this activity. We identifed the RNA binding protein
Bicaudal C (Bicc1) as the crucial post-transcriptional regulator of dand5 (Maerker et al., 2020). To date, a variety of
Bicc1 functions have been reported, ranging from repression
to protection of mRNAs (Rothé et al., 2015; Tran et al., 2010;
Zhang et al., 2014, 2013). Interestingly, Bicc1 was already
implicated in LR asymmetry in mouse and frog because of
its requirement to polarize fow-generating LRO cilia via
planar cell polarity (Maisonneuve et al., 2009). However,
bicc1 is co-expressed with dand5 and nodal in sLRO cells
(Maisonneuve et al., 2009) and inhibits the translation of a
reporter mRNA containing the dand5 3’UTR ( Zhang et al.,
2013). We recently showed that 139 nucleotides of the most
proximal dand5 3’UTR were suffcient to mediate Bicc1
regulation; we termed the sequence the Bicc1 responsive
element (Bicc1RE; Maerker et al., 2020). In vivo, a target
protector MO (tpMO), which blocks accessibility to the
Bicc1RE, prevented left-sided Nodal cascade induction, suggesting that Bicc1 mediates dand5 repression. Remarkably,
f ow-dependent decay of dand5 mRNA was not affected by
the tpMO, strongly suggesting that translation inhibition is
central to Bicc1 function. We postulate that f ow-induced
signaling triggers a yet-unknown posttranslational modifcation of Bicc1 that switches its activity towards translational repression of dand5 (Figure 15.2; Maerker et al.,
2020). Interestingly, we demonstrated that Dicer, a ribonuclease that is critical for miR processing, was expressed
in sLRO cells and is required for f ow-induced dand5
repression. In agreement, dicer morphants lack asymmetric
Nodal cascade induction. We further showed that Bicc1 and
Dicer acted in a synergistic manner, suggesting that both
post-transcriptional mechanisms co-operate during symmetry breakage (Maerker et al., 2020). Taken together, our
Xenopus work provides a new framework for how symmetry breakage is implemented from cilia-generated leftward
f uid fow to the asymmetric release of the Nodal morphogen. Surprisingly, post-transcriptional regulation is the initial target of fow, which opens a new route for sophisticated
analysis in the future.