94
M. Maden
Where Is RA Produced?
raldh2 is expressed in the involuting mesoderm during gastrulation and in the somites
and lateral plate mesoderm after gastrulation (Begemann et al. 2001; Grandell et al.
Grandel et al. 2002). Later, when the fin bud appears, raldh2 is also expressed in the
posterior mesenchyme. Then, it spreads throughout the fin bud and finally regresses
to the proximal anterior fin mesenchyme (Gibert et al. 2006).
The specific somites that produce RA are somites 6–8. Upon ablation of the
somitic mesoderm in embryos with double mutations for spadetail (spt) and no tail
(ntl), somites do not form, there is no tbx5.1 expression, and no pectoral fins.
Remarkably, RA application to these mutants after gastrulation rescued tbx5.1
expression and fin development and transplantation of wild-type cells into the anterior
somites of neckless mutants also rescues the appearance of fin buds (Gibert et al.
2006). All these observations demonstrate that RA is the sole somite-derived initiator
of pectoral fin induction.
In contrast, cyp26a1, a cytochrome enzyme that catalyzes RA degradation, is
expressed in a small region at the base of the pectoral fin bud (Dobbs-McAuliffe
et al. 2004). Surprisingly, disruption of cyp26a1 expression in a gir mutant resulted
in the failure of fin bud formation (Emoto et al. 2005), in the inhibition of tbx5.1 and
shh expression and in the expansion of raldh2 expression to the posterior fin bud
mesenchyme. As the gir mutant can be rescued by injection of morpholinos against
raldh2, it has been suggested that the gir phenotype is caused by an overproduction
of RA, and further, that oppositional expression of RALDH2 and at least one of the
cytochromes optimally regulates RA availability during fin development.
Is There a RA Gradient?
Several transgenic RA reporter zebrafish lines have been created using (1) RAR
response element (RARE) constructs that drive the expression of fluorescent proteins
(Perez-Edwards et al. 2001; White et al. 2007; Waxman and Yelon 2011), (2) a RAR
ligand binding domain fused to the Gal4 DNA binding domain (Mandal et al. 2013),
or (3) activated RARs that fluoresce in the presence of RA (Shimozono et al. 2013).
While these constructs have been useful in demonstrating gradients of RA activity in
the hindbrain and at the posterior end of the embryo (White et al. 2007; Shimozono
et al. 2013), they failed to demonstrate any RA gradient in the fin bud. Other studies
carried out with a raldh2-GFP reporter have shown uniform expression of RALDH2
throughout the pectoral fin bud mesenchyme (Pittlik and Begemann 2012). Thus,
RA is present throughout the fin bud mesenchyme, but whether this is manifested
by a gradient of distribution in the developing fin bud has not been confirmed by
available methods, possibly because the fin bud is so small.
M. Maden
Where Is RA Produced?
raldh2 is expressed in the involuting mesoderm during gastrulation and in the somites
and lateral plate mesoderm after gastrulation (Begemann et al. 2001; Grandell et al.
Grandel et al. 2002). Later, when the fin bud appears, raldh2 is also expressed in the
posterior mesenchyme. Then, it spreads throughout the fin bud and finally regresses
to the proximal anterior fin mesenchyme (Gibert et al. 2006).
The specific somites that produce RA are somites 6–8. Upon ablation of the
somitic mesoderm in embryos with double mutations for spadetail (spt) and no tail
(ntl), somites do not form, there is no tbx5.1 expression, and no pectoral fins.
Remarkably, RA application to these mutants after gastrulation rescued tbx5.1
expression and fin development and transplantation of wild-type cells into the anterior
somites of neckless mutants also rescues the appearance of fin buds (Gibert et al.
2006). All these observations demonstrate that RA is the sole somite-derived initiator
of pectoral fin induction.
In contrast, cyp26a1, a cytochrome enzyme that catalyzes RA degradation, is
expressed in a small region at the base of the pectoral fin bud (Dobbs-McAuliffe
et al. 2004). Surprisingly, disruption of cyp26a1 expression in a gir mutant resulted
in the failure of fin bud formation (Emoto et al. 2005), in the inhibition of tbx5.1 and
shh expression and in the expansion of raldh2 expression to the posterior fin bud
mesenchyme. As the gir mutant can be rescued by injection of morpholinos against
raldh2, it has been suggested that the gir phenotype is caused by an overproduction
of RA, and further, that oppositional expression of RALDH2 and at least one of the
cytochromes optimally regulates RA availability during fin development.
Is There a RA Gradient?
Several transgenic RA reporter zebrafish lines have been created using (1) RAR
response element (RARE) constructs that drive the expression of fluorescent proteins
(Perez-Edwards et al. 2001; White et al. 2007; Waxman and Yelon 2011), (2) a RAR
ligand binding domain fused to the Gal4 DNA binding domain (Mandal et al. 2013),
or (3) activated RARs that fluoresce in the presence of RA (Shimozono et al. 2013).
While these constructs have been useful in demonstrating gradients of RA activity in
the hindbrain and at the posterior end of the embryo (White et al. 2007; Shimozono
et al. 2013), they failed to demonstrate any RA gradient in the fin bud. Other studies
carried out with a raldh2-GFP reporter have shown uniform expression of RALDH2
throughout the pectoral fin bud mesenchyme (Pittlik and Begemann 2012). Thus,
RA is present throughout the fin bud mesenchyme, but whether this is manifested
by a gradient of distribution in the developing fin bud has not been confirmed by
available methods, possibly because the fin bud is so small.