4 RA Signaling in Limb Development and Regeneration …
93
Mutations that inactivate retinaldehyde dehydrogenase type 2 (RALDH2), an
enzyme that catalyzes the last step of RA biosynthesis in vertebrates, produced mutant
zebrafish lacking pectoral fin buds. Interestingly, these mutants (no-fin, Grandel et al.
2002, and neckless, Begemann et al. 2001) were rescued by exogenous administration
of RA. Morpholinos against raldh2, as well as competitive reversible inhibitors of
RALDHs, such as citral or diethylaminobenzaldehyde (DEAB), that disrupt RA
synthesis, also inhibited the development of pectoral fin buds.
Note that raldh2 disruption or inhibition prevented the expression of critical genes,
such as tbx5.1 (the earliest marker of pectoral fin bud development in the lateral plate
mesoderm), shh (a later marker of posterior fin mesenchyme) (Begemann et al. 2001),
wnt2b (expressed in the intermediate mesenchyme between the somites and lateral
plate mesenchyme) (Mercarder et al. 2006), and fibin (expressed in the somites and
lateral plate that abut fin forming mesenchyme) (Wakahara et al. 2007). Interestingly,
embryos treated with fibin morpholinos did not express tbx5.1, suggesting an RAinduced cascade where RA induces fibin, which then regulates the expression of
tbx5.1. Downstream of tbx5.1 are several fgfs and a transcriptional repressor, prdm1
also known as blimp1 (Mercarder et al. 2006).
In zebrafish there are four nuclear retinoic acid receptors (raraa, rarab, rarga and
rargb). The use of morpholinos against these RARs, individually or in combination
revealed that the RA signal for pectoral fin development is transduced only by rarab
(Linville et al. 2009). Indeed, a morpholino against rarab alone, inhibited tbx5.1
expression and pectoral fins development.
When RA signaling is inhibited later, but still at pre-budding stages, by DEAB
or pan RAR antagonists, the resulting fin bud is smaller than normal, and its pattern
is anteriorized. Under these conditions, posteriorizing genes, such as the bHLH
transcription factor, hand2, and sonic hedgehog, shh, which colocalizes with ZPA, are
not expressed (Gibert et al. 2006). Other posteriorizing genes, hoxd11a and hoxd12a,
whose expression do not depend on shh, are also not expressed. Instead, hoxc6a, a
marker of the anterior half of the mesenchyme, is expanded all across the fin bud.
These results suggest that, during the early initiation phase, RA acts in the anterior
somites. Then RA induces the establishment of posterior fates involving shh and
posteriorizing hox genes.
Although this somite-based RA cascade explains pectoral fin initiation and development from the end of gastrulation onwards, it has been noted that administration of
an RAR antagonist at early gastrulation stages generates embryos with an increased
number of precardiac mesoderm cells and a correspondingly decreased number of
other mesodermal lineages including the pancreas, pharyngeal pouches, and pectoral
fin (Keegan et al. 2005). Thus, RA generated at early gastrulation stages could play
an even earlier axial role in establishing the domain sizes of mesodermal progenitors
without which there would not even be a precursor pool of pre-limb bud cells for
somite RA to act on. The balancing molecule in this pool size determination event
is likely to be fibroblast growth factor (FGF) because the pectoral fin deletion in
neckless mutants (which have too many precardiac mesoderm cells) can be rescued
by the abrogation of FGF signaling (Cunningham et al. 2013).
93
Mutations that inactivate retinaldehyde dehydrogenase type 2 (RALDH2), an
enzyme that catalyzes the last step of RA biosynthesis in vertebrates, produced mutant
zebrafish lacking pectoral fin buds. Interestingly, these mutants (no-fin, Grandel et al.
2002, and neckless, Begemann et al. 2001) were rescued by exogenous administration
of RA. Morpholinos against raldh2, as well as competitive reversible inhibitors of
RALDHs, such as citral or diethylaminobenzaldehyde (DEAB), that disrupt RA
synthesis, also inhibited the development of pectoral fin buds.
Note that raldh2 disruption or inhibition prevented the expression of critical genes,
such as tbx5.1 (the earliest marker of pectoral fin bud development in the lateral plate
mesoderm), shh (a later marker of posterior fin mesenchyme) (Begemann et al. 2001),
wnt2b (expressed in the intermediate mesenchyme between the somites and lateral
plate mesenchyme) (Mercarder et al. 2006), and fibin (expressed in the somites and
lateral plate that abut fin forming mesenchyme) (Wakahara et al. 2007). Interestingly,
embryos treated with fibin morpholinos did not express tbx5.1, suggesting an RAinduced cascade where RA induces fibin, which then regulates the expression of
tbx5.1. Downstream of tbx5.1 are several fgfs and a transcriptional repressor, prdm1
also known as blimp1 (Mercarder et al. 2006).
In zebrafish there are four nuclear retinoic acid receptors (raraa, rarab, rarga and
rargb). The use of morpholinos against these RARs, individually or in combination
revealed that the RA signal for pectoral fin development is transduced only by rarab
(Linville et al. 2009). Indeed, a morpholino against rarab alone, inhibited tbx5.1
expression and pectoral fins development.
When RA signaling is inhibited later, but still at pre-budding stages, by DEAB
or pan RAR antagonists, the resulting fin bud is smaller than normal, and its pattern
is anteriorized. Under these conditions, posteriorizing genes, such as the bHLH
transcription factor, hand2, and sonic hedgehog, shh, which colocalizes with ZPA, are
not expressed (Gibert et al. 2006). Other posteriorizing genes, hoxd11a and hoxd12a,
whose expression do not depend on shh, are also not expressed. Instead, hoxc6a, a
marker of the anterior half of the mesenchyme, is expanded all across the fin bud.
These results suggest that, during the early initiation phase, RA acts in the anterior
somites. Then RA induces the establishment of posterior fates involving shh and
posteriorizing hox genes.
Although this somite-based RA cascade explains pectoral fin initiation and development from the end of gastrulation onwards, it has been noted that administration of
an RAR antagonist at early gastrulation stages generates embryos with an increased
number of precardiac mesoderm cells and a correspondingly decreased number of
other mesodermal lineages including the pancreas, pharyngeal pouches, and pectoral
fin (Keegan et al. 2005). Thus, RA generated at early gastrulation stages could play
an even earlier axial role in establishing the domain sizes of mesodermal progenitors
without which there would not even be a precursor pool of pre-limb bud cells for
somite RA to act on. The balancing molecule in this pool size determination event
is likely to be fibroblast growth factor (FGF) because the pectoral fin deletion in
neckless mutants (which have too many precardiac mesoderm cells) can be rescued
by the abrogation of FGF signaling (Cunningham et al. 2013).