100
M. Maden
blastema (Brockes 1992). Similarly, when co-cultured with cells transfected with an
RA-responsive reporter gene, a wound epidermis from a proximal newt blastema
showed higher RA-signaling activity than a wound epidermis from a distal blastema
(Viviano et al. 1995).
Interestingly, upon limb amputation of a RARE-EGFP transgenic axolotl, the
wound epidermis displayed a GFP signal and not the blastema (Monaghan and Maden
2012) showing striking similarity to the newt reporter experiments.
Where Is RA Generated?
Available evidence suggests that RA synthesizing enzymes are likely present in the
apical cap, but this is an uninvestigated area, with the exception of a microarray
analysis that found four genes in the RA pathway, namely raldh3, crabp1, crabp2
and rdh10, were up-regulated in the wound epithelium of the axolotl after limb
amputation (Monaghan et al. 2012).
The Effects of Exogenous RA
The axolotl Ambystoma mexicanum (Fig. 4.2) as well as other species of amphibians
that have been used for limb regeneration studies show proximalization of the limb
by RA (Thoms and Stocum 1984, Niazi et al. 1985; Lheureux et al. 1986; Ju and
Kim 1994). Urodeles (both newt and axolotl) duplicate the PD axis after amputation
and RA treatment unless further surgery is done. However, Anurans (Rana, Xenopus
and Bufo) show an additional effect on the AP axis and can generate complete pairs
of hindlimbs in double posterior orientation even including the pelvic girdle from
foot amputation levels (Fig. 4.3b, Niazi and Saxena 1978; Maden 1983b; Scadding
and Maden 1986a). This phenomenon has been seen in Xenopus after treatment
with a RARβ agonist, or after administration of a CYP26 inhibitor (Cuervo and
Chimal-Monroy 1886), confirming that RA is an endogenous component of the
regenerate.
This remarkable ectopic production of hindlimbs was also seen when the tails
of certain species of frogs are amputated and treated with RA. Then, instead of
regenerating the tail, complete pairs of hindlimbs, including the pelvic girdles are
produced (Fig. 4.3c, Mohanty-Hejmadi et al. 1992; Maden 1993). This effect is
similar to the production of ectopic limbs in mouse embryos and reminds us that
when extra limbs are generated, we cannot distinguish between effects on individual
axes because if one axis is altered, the other two are also altered. In Urodeles, the
effects of RA on the other axes are seen after surgical construction of limbs—double
anterior, double posterior, double dorsal and double ventral limbs. After amputation,
RA posteriorizes and also ventralizes the regenerating limb, that is, all three axes of
the limb are respecified by RA (Stocum and Thoms 1984; Ludolph et al. 1990; Kim
and Stocum 1990; Wigmore 1990).
M. Maden
blastema (Brockes 1992). Similarly, when co-cultured with cells transfected with an
RA-responsive reporter gene, a wound epidermis from a proximal newt blastema
showed higher RA-signaling activity than a wound epidermis from a distal blastema
(Viviano et al. 1995).
Interestingly, upon limb amputation of a RARE-EGFP transgenic axolotl, the
wound epidermis displayed a GFP signal and not the blastema (Monaghan and Maden
2012) showing striking similarity to the newt reporter experiments.
Where Is RA Generated?
Available evidence suggests that RA synthesizing enzymes are likely present in the
apical cap, but this is an uninvestigated area, with the exception of a microarray
analysis that found four genes in the RA pathway, namely raldh3, crabp1, crabp2
and rdh10, were up-regulated in the wound epithelium of the axolotl after limb
amputation (Monaghan et al. 2012).
The Effects of Exogenous RA
The axolotl Ambystoma mexicanum (Fig. 4.2) as well as other species of amphibians
that have been used for limb regeneration studies show proximalization of the limb
by RA (Thoms and Stocum 1984, Niazi et al. 1985; Lheureux et al. 1986; Ju and
Kim 1994). Urodeles (both newt and axolotl) duplicate the PD axis after amputation
and RA treatment unless further surgery is done. However, Anurans (Rana, Xenopus
and Bufo) show an additional effect on the AP axis and can generate complete pairs
of hindlimbs in double posterior orientation even including the pelvic girdle from
foot amputation levels (Fig. 4.3b, Niazi and Saxena 1978; Maden 1983b; Scadding
and Maden 1986a). This phenomenon has been seen in Xenopus after treatment
with a RARβ agonist, or after administration of a CYP26 inhibitor (Cuervo and
Chimal-Monroy 1886), confirming that RA is an endogenous component of the
regenerate.
This remarkable ectopic production of hindlimbs was also seen when the tails
of certain species of frogs are amputated and treated with RA. Then, instead of
regenerating the tail, complete pairs of hindlimbs, including the pelvic girdles are
produced (Fig. 4.3c, Mohanty-Hejmadi et al. 1992; Maden 1993). This effect is
similar to the production of ectopic limbs in mouse embryos and reminds us that
when extra limbs are generated, we cannot distinguish between effects on individual
axes because if one axis is altered, the other two are also altered. In Urodeles, the
effects of RA on the other axes are seen after surgical construction of limbs—double
anterior, double posterior, double dorsal and double ventral limbs. After amputation,
RA posteriorizes and also ventralizes the regenerating limb, that is, all three axes of
the limb are respecified by RA (Stocum and Thoms 1984; Ludolph et al. 1990; Kim
and Stocum 1990; Wigmore 1990).