4 RA Signaling in Limb Development and Regeneration …
103
bud, reflecting increased RA concentrations in this region. In the early limb bud,
RARβ2 transcripts were also detected in proximal mesenchyme, thus indicating AP
and PD differences in RA concentrations.
It must be stressed that chick limb buds contain not only tRA but also 3,4didehydroretinoic acid (ddRA) at a concentration 6 times greater than tRA (Thaller
and Eichele 1990), which means that the total effective concentration of RA is much
higher than 50 nM. Both RA and ddRA can be generated in the limb bud from retinol
(Thaller and Eichele 1988). Further work investigating retinoid concentration differences in the AP axis (Scott et al. 1994) showed that retinol is equally distributed
across the chick limb bud, while RA levels are 40% higher in the posterior bud than
in the anterior side. Synthesis studies (Maden et al. 1998; Stratford et al. 1996; Helms
et al. 1994) showed that limb buds can synthesize both tRA and ddRA from retinol
(although at far lower levels than other regions of the embryo), and that ddRA is synthesized in the posterior half of the limb bud at small, but reproducibly higher levels
(25–40%), than in the anterior one. Interestingly, Maden et al. (1998) also looked at
other regions of the embryo and found there was a 3-fold higher level of total RA
(tRA plus ddRA) in the adjacent somites than in the limb bud itself, supporting the
idea that RA diffuses into the limb bud from the somites where it is synthesised. In
fact, in the embryo, the somites have the highest levels of RA and the distribution of
endogenous retinoids throughout the embryo correlates perfectly with the expression
of raldh2.
Where Is RA Generated?
Strinkingly, no raldhs were found in the chick limb bud at outgrowth stages. Instead
raldh2 was present in the lateral mesoderm from gastrulation stages onwards, and
then, in the somites as they form in a rostral to caudal direction. Up to stage 14,
raldh2 is expressed strongly in the presumptive wing field and leg field lateral plate
mesoderm, but at stage 15, as soon as the limb buds appear, raldh2 is down-regulated.
So as the forelimb grows, the lateral plate is devoid of raldh2. Instead raldh2 and
retinol dehydrogenase (rdh10) were found to be expressed in the coelomic epithelium
at the proximal base of the limb bud (Swindell et al. 1999; Berggren et al. 1999;
Blentic et al. 2003; Reijntjes et al. 2010).
Interestingly, two enzymes that metabolize RA, cyp26a1 and cyp23b, are
expressed in the ectoderm covering the early wing and leg bud (Swindell et al. 1999;
Blentic et al. 2003) and throughout the distal mesenchyme, respectively (Reijntjes
et al. 2003). Therefore, the mesenchyme of the growing limb bud tip is devoid of
RA. Since RA is generated from the adjacent somites, lateral plate, and coelomic
epithelium, there would be high levels of RA only in the proximal mesenchyme
where there is no cyp26 expression. Thus, a potential for generating an RA gradient
in the proximal region exists.
103
bud, reflecting increased RA concentrations in this region. In the early limb bud,
RARβ2 transcripts were also detected in proximal mesenchyme, thus indicating AP
and PD differences in RA concentrations.
It must be stressed that chick limb buds contain not only tRA but also 3,4didehydroretinoic acid (ddRA) at a concentration 6 times greater than tRA (Thaller
and Eichele 1990), which means that the total effective concentration of RA is much
higher than 50 nM. Both RA and ddRA can be generated in the limb bud from retinol
(Thaller and Eichele 1988). Further work investigating retinoid concentration differences in the AP axis (Scott et al. 1994) showed that retinol is equally distributed
across the chick limb bud, while RA levels are 40% higher in the posterior bud than
in the anterior side. Synthesis studies (Maden et al. 1998; Stratford et al. 1996; Helms
et al. 1994) showed that limb buds can synthesize both tRA and ddRA from retinol
(although at far lower levels than other regions of the embryo), and that ddRA is synthesized in the posterior half of the limb bud at small, but reproducibly higher levels
(25–40%), than in the anterior one. Interestingly, Maden et al. (1998) also looked at
other regions of the embryo and found there was a 3-fold higher level of total RA
(tRA plus ddRA) in the adjacent somites than in the limb bud itself, supporting the
idea that RA diffuses into the limb bud from the somites where it is synthesised. In
fact, in the embryo, the somites have the highest levels of RA and the distribution of
endogenous retinoids throughout the embryo correlates perfectly with the expression
of raldh2.
Where Is RA Generated?
Strinkingly, no raldhs were found in the chick limb bud at outgrowth stages. Instead
raldh2 was present in the lateral mesoderm from gastrulation stages onwards, and
then, in the somites as they form in a rostral to caudal direction. Up to stage 14,
raldh2 is expressed strongly in the presumptive wing field and leg field lateral plate
mesoderm, but at stage 15, as soon as the limb buds appear, raldh2 is down-regulated.
So as the forelimb grows, the lateral plate is devoid of raldh2. Instead raldh2 and
retinol dehydrogenase (rdh10) were found to be expressed in the coelomic epithelium
at the proximal base of the limb bud (Swindell et al. 1999; Berggren et al. 1999;
Blentic et al. 2003; Reijntjes et al. 2010).
Interestingly, two enzymes that metabolize RA, cyp26a1 and cyp23b, are
expressed in the ectoderm covering the early wing and leg bud (Swindell et al. 1999;
Blentic et al. 2003) and throughout the distal mesenchyme, respectively (Reijntjes
et al. 2003). Therefore, the mesenchyme of the growing limb bud tip is devoid of
RA. Since RA is generated from the adjacent somites, lateral plate, and coelomic
epithelium, there would be high levels of RA only in the proximal mesenchyme
where there is no cyp26 expression. Thus, a potential for generating an RA gradient
in the proximal region exists.