116
Xenopus
a segmental boundary (Kim et al., 2000). ripply2.2, which
encodes a WRPW-containing protein, is also required for
the formation of somite boundaries. While ripply2.2 is
required for dll1 and hes4 expression in their proper position in the presomitic mesoderm (Kondow et al., 2007)
(Table 7.12), Notch/RBPJ signaling localizes ripply2.2 in
the anterior halves of somitomeres (Kondow et al., 2006)
(Figure 7.1D), which in turn further restricts dlc to the anterior border of the most anterior somitomeres by recruiting
the TLE4 co-repressor (Kondow et al., 2006). It was proposed that Tbx6 acts as a transcriptional activator of segmental target genes in posterior somitomeres but changes to
a transcriptional repressor through binding to a Ripply2.2/
TLE4 complex when Ripply2.2 accumulates above a threshold level in the most anterior somitomeres. In this way,
Ripply2.2 and Tbx6 contribute to terminate the segmentation program in the anterior presomitic mesoderm (Kondow
et al., 2007; Hitachi et al., 2008). Interestingly, the related
ripply2.1, whose striped expression in the anterior presomitic mesoderm is also dependent on Notch/RBPJ signaling
(Figure 7.1D) (Table 7.12), is not required for segmentation
but for positioning the segmentation front via RA signaling
(Chan et al., 2006). The Dlc/Notch/RBPJ pathway is also
necessary for striped rnd1 and rnd3 expression in somitomeres (Figure 7.1D) (Table 7.12). They encode GTP binding
proteins required for segmentation independently of hes7.3/
esr5. rnd1 is expressed in the anterior half of somitomeres,
whereas rnd3 is expressed in the boundary between the
anterior and posterior halves, suggesting different roles in
segmentation (Goda et al., 2009).
hox genes are necessary for somitogenesis and establish a reciprocal positive regulation with dlc in the paraxial
mesoderm (Tables 7.15, 7.12). dlc might regulate the timer
for temporal collinearity of hox gene expression, which in
turn controls somite anterior-posterior identity (Peres et al.,
2006; Durston et al., 2012).
The Notch pathway also is regulated during somitogenesis at other levels. For example, celf1, which encodes
an RNA-binding protein that mediates sequence-specif c
mRNA deadenylation, binds the 3’UTR of RBPJ mRNA
promoting its degradation; this is required to control the
interplay between FGF and RA signaling that governs the
determination front (Gautier-Courteille et al., 2004; Cibois
et al., 2010; Cibois et al., 2013). RA treatments or FGF pathway blockade repressed dlc, hes5.6, hes6.1, and hes7.3/
esr5 in the tailbud, shifting their expression domain caudally (Moreno and Kintner, 2004). RARβ2 knock-down
reduced somite number, increased somite size, and rostrally
expanded presomitic mesoderm markers, including hes7.3/
esr5. Microarray analysis showed that manipulating RA signaling signifcantly changed hes5.3 levels (Janesick et al.,
2017 ) ( Table 7.12 ).
7.4. FUTURE DIRECTIONS
Work in Xenopus frequently has led the feld in addressing the role of notch1 and dll1 in several developmental
programs, as well as dlc in somitogenesis, but there is much
less information about other Notch receptors and ligands
during frog development. Expression patterns for some are
available in limited types of tissues. For example, notch2 is
expressed in the PPE and during lens development, where it
is positively controlled by hes4 (Ogino et al., 2008; Murato
and Hashimoto, 2009); notch1, notch2, jag1, and jag2 are
expressed in the liver during metamorphosis (Ueno et al.,
2015), whereas notch4 and dll4 are implicated in the arterial endothelial program (Ciau-Uitz et al., 2010; Leung et al.,
2013; Nimmo et al., 2013; Kirmizitas et al., 2017). RNAseq
data (Session et al., 2016) (Figure 7.1C, right column) suggest that notch3, jag1, jag2, and dll4 might have roles during
embryogenesis, but we need to know their spatial distributions at different developmental timepoints. Finally, since
the notch4 gene model was not available at the time of the
RNAseq study, a developmental expression profle is not yet
available.
To dissect Notch signaling involvement in developmental
processes more precisely, a combination of strategies will be
necessary. For example, experiments employing RBPJ DBM,
psen MO, or γ-Secretase inhibitors impair the function of
every notch paralogue; knock-down/knock-out strategies
are needed to provide results specifc for each paralogue.
Strikingly, only notch1 has been knocked down so far, both in
studies by our group concerning germ layers, DML, and DV
axis, and by others concerning ciliogenesis in the epidermis,
GRP and left-right patterning (Sakano et al., 2010; Tözser
et al., 2015; Tomankova et al., 2017). Since there is growing
evidence of RBPJ-independent Ligand/Notch functions in
several biological contexts (Hayward et al., 2005), including various aspects of Xenopus development (Revinski et al.,
2010; Peres et al., 2006; Acosta et al., 2011), knock-down/
knock-out approaches next need to address both canonical
and non-canonical functions. As RBPJ has dual properties,
activating or repressing Notch-targets depending on the
ON/OFF status of Notch signaling, it might not always be
straightforward to interpret the results of RBPJ blockade in
complex contexts, including those in which multiple inputs
from different ligands might take place. For example, in
some studies, RBPJ DBM produced milder or more variable
effects in comparison to the blockade of one ligand or the
receptor (Revinski et al., 2010) or did not affect the process
under study (Takada et al., 2005; Peres et al., 2006; Nichane
et al., 2008a). The effects of protecting RBPJ mRNA from
Celf1-mediated degradation were compatible with a Notch/
RBPJ gain-of-function in the posterior presomitic mesoderm and with a Notch/RBPJ loss-of-function in the anterior
presomitic mesoderm (Cibois et al., 2013). In addition, the
response to perturbing Notch signaling can change abruptly
at certain developmental transitions (Contakos et al., 2005;
Revinski et al., 2010), thus requiring a more detailed analysis of gene markers and phenotypes by time-controlled
manipulations.
Work from different animal models and cell types show
that the number of direct Notch/RBPJ targets outside the
hes/hey families is constantly growing, including genes
Xenopus
a segmental boundary (Kim et al., 2000). ripply2.2, which
encodes a WRPW-containing protein, is also required for
the formation of somite boundaries. While ripply2.2 is
required for dll1 and hes4 expression in their proper position in the presomitic mesoderm (Kondow et al., 2007)
(Table 7.12), Notch/RBPJ signaling localizes ripply2.2 in
the anterior halves of somitomeres (Kondow et al., 2006)
(Figure 7.1D), which in turn further restricts dlc to the anterior border of the most anterior somitomeres by recruiting
the TLE4 co-repressor (Kondow et al., 2006). It was proposed that Tbx6 acts as a transcriptional activator of segmental target genes in posterior somitomeres but changes to
a transcriptional repressor through binding to a Ripply2.2/
TLE4 complex when Ripply2.2 accumulates above a threshold level in the most anterior somitomeres. In this way,
Ripply2.2 and Tbx6 contribute to terminate the segmentation program in the anterior presomitic mesoderm (Kondow
et al., 2007; Hitachi et al., 2008). Interestingly, the related
ripply2.1, whose striped expression in the anterior presomitic mesoderm is also dependent on Notch/RBPJ signaling
(Figure 7.1D) (Table 7.12), is not required for segmentation
but for positioning the segmentation front via RA signaling
(Chan et al., 2006). The Dlc/Notch/RBPJ pathway is also
necessary for striped rnd1 and rnd3 expression in somitomeres (Figure 7.1D) (Table 7.12). They encode GTP binding
proteins required for segmentation independently of hes7.3/
esr5. rnd1 is expressed in the anterior half of somitomeres,
whereas rnd3 is expressed in the boundary between the
anterior and posterior halves, suggesting different roles in
segmentation (Goda et al., 2009).
hox genes are necessary for somitogenesis and establish a reciprocal positive regulation with dlc in the paraxial
mesoderm (Tables 7.15, 7.12). dlc might regulate the timer
for temporal collinearity of hox gene expression, which in
turn controls somite anterior-posterior identity (Peres et al.,
2006; Durston et al., 2012).
The Notch pathway also is regulated during somitogenesis at other levels. For example, celf1, which encodes
an RNA-binding protein that mediates sequence-specif c
mRNA deadenylation, binds the 3’UTR of RBPJ mRNA
promoting its degradation; this is required to control the
interplay between FGF and RA signaling that governs the
determination front (Gautier-Courteille et al., 2004; Cibois
et al., 2010; Cibois et al., 2013). RA treatments or FGF pathway blockade repressed dlc, hes5.6, hes6.1, and hes7.3/
esr5 in the tailbud, shifting their expression domain caudally (Moreno and Kintner, 2004). RARβ2 knock-down
reduced somite number, increased somite size, and rostrally
expanded presomitic mesoderm markers, including hes7.3/
esr5. Microarray analysis showed that manipulating RA signaling signifcantly changed hes5.3 levels (Janesick et al.,
2017 ) ( Table 7.12 ).
7.4. FUTURE DIRECTIONS
Work in Xenopus frequently has led the feld in addressing the role of notch1 and dll1 in several developmental
programs, as well as dlc in somitogenesis, but there is much
less information about other Notch receptors and ligands
during frog development. Expression patterns for some are
available in limited types of tissues. For example, notch2 is
expressed in the PPE and during lens development, where it
is positively controlled by hes4 (Ogino et al., 2008; Murato
and Hashimoto, 2009); notch1, notch2, jag1, and jag2 are
expressed in the liver during metamorphosis (Ueno et al.,
2015), whereas notch4 and dll4 are implicated in the arterial endothelial program (Ciau-Uitz et al., 2010; Leung et al.,
2013; Nimmo et al., 2013; Kirmizitas et al., 2017). RNAseq
data (Session et al., 2016) (Figure 7.1C, right column) suggest that notch3, jag1, jag2, and dll4 might have roles during
embryogenesis, but we need to know their spatial distributions at different developmental timepoints. Finally, since
the notch4 gene model was not available at the time of the
RNAseq study, a developmental expression profle is not yet
available.
To dissect Notch signaling involvement in developmental
processes more precisely, a combination of strategies will be
necessary. For example, experiments employing RBPJ DBM,
psen MO, or γ-Secretase inhibitors impair the function of
every notch paralogue; knock-down/knock-out strategies
are needed to provide results specifc for each paralogue.
Strikingly, only notch1 has been knocked down so far, both in
studies by our group concerning germ layers, DML, and DV
axis, and by others concerning ciliogenesis in the epidermis,
GRP and left-right patterning (Sakano et al., 2010; Tözser
et al., 2015; Tomankova et al., 2017). Since there is growing
evidence of RBPJ-independent Ligand/Notch functions in
several biological contexts (Hayward et al., 2005), including various aspects of Xenopus development (Revinski et al.,
2010; Peres et al., 2006; Acosta et al., 2011), knock-down/
knock-out approaches next need to address both canonical
and non-canonical functions. As RBPJ has dual properties,
activating or repressing Notch-targets depending on the
ON/OFF status of Notch signaling, it might not always be
straightforward to interpret the results of RBPJ blockade in
complex contexts, including those in which multiple inputs
from different ligands might take place. For example, in
some studies, RBPJ DBM produced milder or more variable
effects in comparison to the blockade of one ligand or the
receptor (Revinski et al., 2010) or did not affect the process
under study (Takada et al., 2005; Peres et al., 2006; Nichane
et al., 2008a). The effects of protecting RBPJ mRNA from
Celf1-mediated degradation were compatible with a Notch/
RBPJ gain-of-function in the posterior presomitic mesoderm and with a Notch/RBPJ loss-of-function in the anterior
presomitic mesoderm (Cibois et al., 2013). In addition, the
response to perturbing Notch signaling can change abruptly
at certain developmental transitions (Contakos et al., 2005;
Revinski et al., 2010), thus requiring a more detailed analysis of gene markers and phenotypes by time-controlled
manipulations.
Work from different animal models and cell types show
that the number of direct Notch/RBPJ targets outside the
hes/hey families is constantly growing, including genes