4 RA Signaling in Limb Development and Regeneration …
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transform into a completely different molecular state when they become regenerating cells such that the same dose of retinoid induces a totally different outcome. It
would be an important advance for teratology and regenerative biology if the molecular events occurring during this cellular transformation were determined. Figure 4.4
summarizes the RA machinery and effects on amphibian limb development.
Amphibian Limb Regeneration
Is RA Required?
After amputation, regeneration is defective or prevented when regenerating axolotl
limbs are treated with disulphiram or citral (Maden 1998; Scadding 1999). The same
results were seen with an RARβ antagonist (Del Rincon and Scadding 2002; Nguyen
et al. 2017). These findings suggest that RA signaling is required for amphibian limb
regeneration.
Studies in the regenerating newt limb revealed there are at least 6 RAR isoforms:
α 1 , α 2 , δ 1a , δ 1b, δ 2 (the newt δ is equivalent to the mammalian γ ) and β 2 , each one
having a different function during regeneration. Domain swap experiments have been
conducted in which the RA-binding domain of each receptor has been replaced with
the ligand binding domain from the thyroid hormone receptor. The DNA-binding
specificity was retained, but the receptors were now responsive to thyroid hormone
instead of RA. Using these chimeric constructs, it has been shown that the RARα1
receptor is responsible for the proliferation of blastemal cells (Schilthius et al. 1993),
the RARδ1 receptor for secretory differentiation in the wound epidermis (Pecorino
et al. 1994), and the RARδ2 isoform for the ability of RA to proximalize the blastema
(Pecorino et al. 1996).
Is RA Present?
RA has been identified in regenerating blastema of amphibians by several methods.
HPLC analysis measured RA concentration in the regenerative limb of the axolotl
Ambystoma mexicanum after radius-ulna or humerus level amputation (Scadding and
Maden 1994). All-trans RA (tRA) levels were five-fold higher in the posterior side
of the regenerative blastema than on the anterior side suggesting the existence of
an anteroposterior gradient. Moreover, 2.7-fold higher levels were found in distal
blastemas compared to proximal blastemas. However, in Xenopus regenerating limb
blastemas, tRA was detected at lower levels than in axolotl and there was no evidence
of any gradient.
Intriguingly, other methods using RA reporter contructs gave different results. In
the context of the Newt, limb cells were transfected with an RA-responsive reporter
construct and injected into proximal or distal blastema. Interestingly, the cells injected
in a proximal blastema were activated 3.5 times more than those injected in a distal
99
transform into a completely different molecular state when they become regenerating cells such that the same dose of retinoid induces a totally different outcome. It
would be an important advance for teratology and regenerative biology if the molecular events occurring during this cellular transformation were determined. Figure 4.4
summarizes the RA machinery and effects on amphibian limb development.
Amphibian Limb Regeneration
Is RA Required?
After amputation, regeneration is defective or prevented when regenerating axolotl
limbs are treated with disulphiram or citral (Maden 1998; Scadding 1999). The same
results were seen with an RARβ antagonist (Del Rincon and Scadding 2002; Nguyen
et al. 2017). These findings suggest that RA signaling is required for amphibian limb
regeneration.
Studies in the regenerating newt limb revealed there are at least 6 RAR isoforms:
α 1 , α 2 , δ 1a , δ 1b, δ 2 (the newt δ is equivalent to the mammalian γ ) and β 2 , each one
having a different function during regeneration. Domain swap experiments have been
conducted in which the RA-binding domain of each receptor has been replaced with
the ligand binding domain from the thyroid hormone receptor. The DNA-binding
specificity was retained, but the receptors were now responsive to thyroid hormone
instead of RA. Using these chimeric constructs, it has been shown that the RARα1
receptor is responsible for the proliferation of blastemal cells (Schilthius et al. 1993),
the RARδ1 receptor for secretory differentiation in the wound epidermis (Pecorino
et al. 1994), and the RARδ2 isoform for the ability of RA to proximalize the blastema
(Pecorino et al. 1996).
Is RA Present?
RA has been identified in regenerating blastema of amphibians by several methods.
HPLC analysis measured RA concentration in the regenerative limb of the axolotl
Ambystoma mexicanum after radius-ulna or humerus level amputation (Scadding and
Maden 1994). All-trans RA (tRA) levels were five-fold higher in the posterior side
of the regenerative blastema than on the anterior side suggesting the existence of
an anteroposterior gradient. Moreover, 2.7-fold higher levels were found in distal
blastemas compared to proximal blastemas. However, in Xenopus regenerating limb
blastemas, tRA was detected at lower levels than in axolotl and there was no evidence
of any gradient.
Intriguingly, other methods using RA reporter contructs gave different results. In
the context of the Newt, limb cells were transfected with an RA-responsive reporter
construct and injected into proximal or distal blastema. Interestingly, the cells injected
in a proximal blastema were activated 3.5 times more than those injected in a distal