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Fig. 1. Expression of X. laevis Rarβ1 and Rarβ2.
(A-C) Whole-mount in situ hybridization of Rarβ1 and Rarβ2
mRNA expression at stage 26 in lateral (A,C) and anterior (B)
views. Rarβ2 is expressed in the hatching gland and mature
somites, with weaker expression in the eye and branchial
arches. Rarβ1 is undetectable by whole-mount in situ
hybridization. (D) QPCR showing Rarβ1 and Rarβ2 gene
expression averaging two biological replicates over
developmental time. Error bars indicate s.e.m. The y-axis
represents 2−δCt values (adjusted for primer efficiency),
normalized to a reference gene Histone H4.
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Fig. 2. Rarβ2 is induced by TTNPB and is regulated by RARα and/or
RARγ. (A) QPCR showing Rarα1, Rarα2, Rarβ2, Rarγ1 and Rarγ2 expression
in embryos treated at stage 7/8 with 1 μM TTNPB, 1 μM AGN193109 or vehicle
(0.1% ethanol) and collected at tailbud stage. The y-axis represents 2−δδCt
values normalized to Eef1a1 and expressed as fold induction relative to control
vehicle (n=5 biological replicates) using standard propagation of error
(Bevington and Robinson, 2003). Error bars indicate s.e.m. An unpaired t-test
in GraphPad Prism v5.0 is reported (*P≤0.05, **P≤0.01, ***P≤0.001).
(B) Embryos were injected unilaterally at the 2- or 4-cell stage with 6.6 ng Rarα
MOs or Rarγ MOs. The injected side is indicated by magenta β-gal lineage
tracer. Rarα MOs and Rarγ MOs knock down the expression of Rarβ2 (α MOs,
13/13 embryos; γ MOs, 8/8) at tailbud stage. Embryos are shown in dorsal view
with anterior on the left. Midline is indicated by a broken green line.
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Fig. 3. Xenopus laevis RARβ2 promoter elements are required for RA responsiveness. Luciferase reporters were selectively mutated for the canonical (C)
direct repeat 5 (DR5) (Sucov et al., 1990), upstream DR5 and upstream half-site (HS). Embryos were injected unilaterally at the 2- or 4-cell stage with 50 pg
reporter DNA then treated at blastula stage with 0.1 μM TTNPB or vehicle (0.1% ethanol). Embryos were collected at neurula stage (each data point represents
one pool of 10 embryos). Data are represented either as relative light units measured by the luminometer or fold induction relative to vehicle using standard
propagation of error (Bevington and Robinson, 2003). TTNPB responsiveness is reduced by mutating either the canonical DR5 or upstream DR5. Both basal
reporter activity and TTNPB responsiveness is reduced by mutating the upstream half-site; however, fold induction is equivalent to wild type. Error bars indicate
s.e.m. An unpaired t-test in GraphPad Prism v5.0 is reported (***P≤0.001; **P≤0.01).
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Fig. 4. Somite morphology and migration of hypaxial
muscle migration are disrupted in Rarβ2 MO-injected
tadpoles. (A-C) Embryos were microinjected bilaterally at the
2-cell stage with 26 ng Rarβ2.L+26 ng Rarβ2.S MOs or 52 ng
control MO. (A,B) Rarβ2 MOs result in paralysis and curved body
axis. Myod marks the somites that are thicker and fewer in
number without v-shape morphology (17/18 embryos)
compared with control MO. Red arrowheads indicate migrating
hypaxial myoblasts in controls, not observed in Rarβ2 MOembryos.
(C) Higher magnification of blurred/disorganized
somite morphologies observed in some embryos marked by
Myod in control and Rarβ2 MO-injected embryos.
(D-G) Embryos were microinjected unilaterally at the 2- or 4-cell
stage with 26 ng Rarβ2.L+26 ng Rarβ2.S MOs. (D,E) The
injected side displays thicker disorganized somites (marked by
Myod) without v-shape morphology (20/21 embryos), compared
with the uninjected side. Red outline indicates melanophores
and migrating hypaxial muscle that are absent on the injected
side. (F,G) The injected side shows diminished hypaxial Tbx3
expression (11/12 embryos), compared with the robust hypaxial
migration on the uninjected side (red arrowheads). All embryos
are shown in lateral view at stage 40; anterior on the left.
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Fig. 5. Somite number is reduced and length increased in
Rarβ2 MO-injected embryos. (A-F) Embryos were
microinjected unilaterally at the 2- or 4-cell stage with 26 ng
Rarβ2.L+26 ng Rarβ2.S MOs or 52 ng control MO. (A,B) Two
lateral sides of the same embryo are shown at stage 26; anterior
on the left. Injected side is indicated by magenta β-gal lineage
tracer. Rarβ2 MOs (B) disrupt and disorganize the chevronshaped
somite morphology, reduce somite number and increase
somite thickness (18/18 embryos) compared with the uninjected
side (A), as indicated by Myod expression. (C-F) Higher
magnification of somite morphologies (marked by Myod) on the
Rarβ2 MO-injected side observed in different embryos.
(G,H) Paraffin wax-embedded coronal sections of embryos from
(A-F). (I) Somite number is quantitated from sectioned embryos;
each data point represents one embryo (n=7). (J) Somite size
(length from posterior to anterior end) is quantitated from
sectioned embryos using ImageJ (units are distance in pixels);
each data point represents one somite. R, rostral somites; C,
caudal somites. Statistics for I,J were calculated in GraphPad
Prism v5 using a t-test (*P≤0.05; ***P≤0.001).
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Fig. 6. Rostral shifting and expansion of somitomere and presomitic
mesoderm markers occurs in Rarβ2 MO-injected embryos. (A-F) Embryos
were injected unilaterally at the 2- or 4-cell stage with 26 ng Rarβ2.L MO+26 ng
Rarβ2.S MO. Injected side is indicated by magenta β-gal lineage tracer.
Neurula stage embryos shown in dorsal view with anterior on the left. Rarβ2
MOs rostrally shift somitomere markers Ripply2 (A) and Mespa/Thyl2 (B), and
thicken their boundaries of expression (Ripply2, 25/27 embryos; Mespa, 26/
31). The expression domains of presomitic mesoderm markers Tbx6 (C),
Msgn1 (D) and Fgf8 (E), and the Notch direct target Esr5 (F) are expanded
rostrally (red vertical lines) by Rarβ2 MOs (Tbx6, 26/28 embryos; Msgn1, 7/9;
Fgf8, 9/13; Esr5, 19/20). Broken red line indicates the midline.
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Fig. 7. Tbx6 and Tbx3 are differentially regulated by
Ripply2 in vivo. Whole-embryo luciferase assay reflecting
Tbx6 or Tbx3 transcriptional activity in the presence or
absence of wild-type or mutant Ripply2 (Bowline or
Ledgerline). Each data point represents one pool of five
embryos, collected from different clutches of females (as
indicated). Error bars indicate s.e.m. One-way ANOVA and
Bonferroni’s multiple comparison test was conducted using
GraphPad Prism: ###P≤0.001 relative to reporter alone;
***P≤0.001 and *P≤0.05 relative to reporter+Tbx6 mRNA.
Tbx6 increases activity ∼3-fold. Ripply2 (Bowline or
Ledgerline) mRNAs repress activity to basal levels when coinjected
with Tbx6 mRNA; microinjection of Ripply2 (Bowline
or Ledgerline) mRNA mutated (Mut) for the WRPW and
FPVQ domain does not repress Tbx6 reporter activity. Tbx3
reduces activity by about 90%, while Ripply2 (Bowline or
Ledgerline or mutants) does not affect Tbx3 reporter activity.
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Fig. 8. Summary of RARβ2 loss-of-function phenotypes and RARβ2-
mediated regulation of Tbx6 and Tbx3 in somitogenesis and hypaxial
myoblast migration. Xenopus Rarβ2 is the RAR subtype most upregulated in
response to ligand. The localization of Rarβ2 in the trunk somites positions it to
respond to RA and control somitogenesis. RARβ2 regulates somite chevron
morphology, restricts the PSM anterior boundary and promotes hypaxial
myoblast migration. RARβ2 loss of function yields fewer and larger somites,
often with disorganized or blurred domains. Ripply2 converts Tbx6 to a
transcriptional repressor in vivo, but does not influence Tbx3 transcriptional
activity. RARβ2 positively regulates Tbx3 to promote hypaxial muscle migration
and negatively regulates Tbx6 to restrict the PSM and caudal progenitor pool.
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supplementary figure 1
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supplementary figure 2
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supplementary figure 3
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supplementary figure 4
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supplementary figure 5
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supplementary figure 6
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supplementary figure 7
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supplementary figure 8
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supplementary figure 9
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supplementary figure S10. Double WISH of RarB2 and Ripply2
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supplementary figure 11
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supplementary figure 12
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supplementary figure 13
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rarb (retinoic acid receptor beta) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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mybpc1 (myosin binding protein C1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up and coronal cross-section
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