98
M. Maden
likely being initiated (Monaghan and Maden 2012). During outgrowth, the forelimbs showed a uniform green fluorescence throughout the mesoderm, suggesting
the presence of RA signaling throughout this region. Thus, these data suggest that
RA is present in the anterior somites and in the developing forelimb. Strikingly, the
hindlimbs did not show any reporter activity.
Where Is RA Generated?
In the developing frog, Eleutherodactylus, there is clear expression of raldh2 in the
anterior somites after gastrulation. Then, when the forelimb bud appears, raldh2 is
expressed at the proximal base in the lateral plate mesoderm, potentially providing
a source of RA for limb outgrowth (Elinson et al. 2008). However, the actual data
concerning raldh2 expression in other species are less clear.
In Xenopus, McEwan et al. showed that, as the limb bud grows out, raldh2 is
expressed throughout the proximal half of the limb bud itself in both forelimbs and
hindlimbs (McEwan et al. 2011), while cyp26b is expressed in the corresponding
distal domain of the limb bud. This could potentially generate a gradient of RA,
with the highest concentration localized proximally. Later, raldh2 was found in the
interdigital mesenchyme along with cyp26a in both fore and hindlimbs (McEwan
et al. 2011). Then as the hindlimbs and forelimbs digits form, raldh2 is limited to
cells surrounding cartilage and proximal expression disappears.
However other studies reported that, in Xenopus, raldh2 is expressed immediately
after gastrulation in the lateral mesoderm and the anterior lateral plate mesoderm,
but disappears as soon as the somites form (Chen et al. 2001; Lynch et al. 2011). In
contrast, in axolotl, raldh2 expression seems to be different, being present certainly
distally and probably throughout the limb bud, thus more resembling the zebrafish
expression pattern.
The Effects of Excess RA
It has been consistently found that administration of excess RA during limb development of axolotl or frog (Xenopus, Scadding and Maden 1986a, b; Alsop et al.
2004; or several species of Rana from North America, Degitz et al. 2003), does not
induce limb duplications. Instead, excess RA has a negative, teratogenic effect that
is concentration and stage dependent and that results in the loss, fusion, or shortening of the long bones of the limb. These effects resemble the classical mammalian
teratogenesis effect that are observed when retinoids are administered to pregnant
mammals (Satre and Kochhar 1989).
However, when the very tip of a limb bud is cut off before application of the
same doses of retinoids, a completely different result is obtained, that is, proximodistal reduplications of limbs. This result is similar to what is observed in mature
regenerating limbs (Scadding and Maden 1986a, b). Thus, the developing cells must
M. Maden
likely being initiated (Monaghan and Maden 2012). During outgrowth, the forelimbs showed a uniform green fluorescence throughout the mesoderm, suggesting
the presence of RA signaling throughout this region. Thus, these data suggest that
RA is present in the anterior somites and in the developing forelimb. Strikingly, the
hindlimbs did not show any reporter activity.
Where Is RA Generated?
In the developing frog, Eleutherodactylus, there is clear expression of raldh2 in the
anterior somites after gastrulation. Then, when the forelimb bud appears, raldh2 is
expressed at the proximal base in the lateral plate mesoderm, potentially providing
a source of RA for limb outgrowth (Elinson et al. 2008). However, the actual data
concerning raldh2 expression in other species are less clear.
In Xenopus, McEwan et al. showed that, as the limb bud grows out, raldh2 is
expressed throughout the proximal half of the limb bud itself in both forelimbs and
hindlimbs (McEwan et al. 2011), while cyp26b is expressed in the corresponding
distal domain of the limb bud. This could potentially generate a gradient of RA,
with the highest concentration localized proximally. Later, raldh2 was found in the
interdigital mesenchyme along with cyp26a in both fore and hindlimbs (McEwan
et al. 2011). Then as the hindlimbs and forelimbs digits form, raldh2 is limited to
cells surrounding cartilage and proximal expression disappears.
However other studies reported that, in Xenopus, raldh2 is expressed immediately
after gastrulation in the lateral mesoderm and the anterior lateral plate mesoderm,
but disappears as soon as the somites form (Chen et al. 2001; Lynch et al. 2011). In
contrast, in axolotl, raldh2 expression seems to be different, being present certainly
distally and probably throughout the limb bud, thus more resembling the zebrafish
expression pattern.
The Effects of Excess RA
It has been consistently found that administration of excess RA during limb development of axolotl or frog (Xenopus, Scadding and Maden 1986a, b; Alsop et al.
2004; or several species of Rana from North America, Degitz et al. 2003), does not
induce limb duplications. Instead, excess RA has a negative, teratogenic effect that
is concentration and stage dependent and that results in the loss, fusion, or shortening of the long bones of the limb. These effects resemble the classical mammalian
teratogenesis effect that are observed when retinoids are administered to pregnant
mammals (Satre and Kochhar 1989).
However, when the very tip of a limb bud is cut off before application of the
same doses of retinoids, a completely different result is obtained, that is, proximodistal reduplications of limbs. This result is similar to what is observed in mature
regenerating limbs (Scadding and Maden 1986a, b). Thus, the developing cells must