4 RA Signaling in Limb Development and Regeneration …
101
In a first attempt to identify the molecules mediating proximalization during limb
regeneration, a differential screen from newt distal limb blastema treated or not with
RA after amputation was performed (Morais Da Silva et al. 2002). A GPI-linked
protein called PROD-1, which is expressed on the cell surface, was first identified in
the newt. It has been suggested that PROD-1 would be implicated in the local cellcell interactions mediating positional identity. The expression of prod-1 in normal
and regenerative limbs is regulated by RA and by proximodistal location. Indeed,
the level of prod-1 was found to be 1.7-fold higher in proximal limb regions than
in distal limb regions (Kumar et al. 2007a, b). When prod-1 is over-expressed in
blastemal cells that normally form distal elements (such as the hand and digits), then
its positional identity is changed to more proximal cells contributing to the formation
of the radius, ulna and tissues extending back to the elbow (Echeverri and Tanaka
2005). Two targets of prod-1 have been identified: MMP-9, which is up-regulated via
the epidermal growth factor (EGF) receptor and provides a link between positional
identity and the extracellular matrix (Blassberg et al. 2010), and nAG, a neurotrophic
factor, (Kumar et al. 2007b), which provides a fascinating link between positional
identity and proliferation.
Interestingly, the prod-1 promoter depicts two binding sites for meis gene products, which are also involved in PD determination during embryonic limb development, suggesting that RA induces similar mechanisms for axial determination during
limb development and regeneration (Shaikh et al. 2011).
Recently, microarray studies performed with RA-treated axolotl distal blastemas
after amputation, revealed gene transcriptional responses associated with RAinduced proximodistal duplications (Nguyen et al. 2017; Voss et al. 2018). At least
100 genes were up-regulated including RA signaling genes (cyp26a1, cyp26B1,
nrip1), several genes normally expressed in proximal developing limb buds in other
vertebrates (meis1, meis2, pbx1, arid5b, fibin„ epha7, fabp2, uroplakin3a, tcaf1,
keratin19, rnd3), and ECM genes (aggrecan and brevican). Conversely, other genes
that are normally expressed in the distal part of the developing limb bud were downregulated, including lhx9, zic5, lmo1, lhx2, spry1, msx2 and hoxa13. Again, the
similarities between genes involved in PD specification in the limb bud and the
limb regenerate are striking. Figure 4.4 shows the distribution of the RA enzymatic
machinery and the effect of an excess or a deficiency of RA on the regenerating
amphibian limb. A summary of the RA-dependent gene cascades is shown in Fig. 4.5.
Chick Limb Development
Is RA Required?
Disulphiram application at chick pre-limb bud or early limb bud stages, abolished
forelimb development as well as shh expression in the ZPA (Stratford et al. 1996).
101
In a first attempt to identify the molecules mediating proximalization during limb
regeneration, a differential screen from newt distal limb blastema treated or not with
RA after amputation was performed (Morais Da Silva et al. 2002). A GPI-linked
protein called PROD-1, which is expressed on the cell surface, was first identified in
the newt. It has been suggested that PROD-1 would be implicated in the local cellcell interactions mediating positional identity. The expression of prod-1 in normal
and regenerative limbs is regulated by RA and by proximodistal location. Indeed,
the level of prod-1 was found to be 1.7-fold higher in proximal limb regions than
in distal limb regions (Kumar et al. 2007a, b). When prod-1 is over-expressed in
blastemal cells that normally form distal elements (such as the hand and digits), then
its positional identity is changed to more proximal cells contributing to the formation
of the radius, ulna and tissues extending back to the elbow (Echeverri and Tanaka
2005). Two targets of prod-1 have been identified: MMP-9, which is up-regulated via
the epidermal growth factor (EGF) receptor and provides a link between positional
identity and the extracellular matrix (Blassberg et al. 2010), and nAG, a neurotrophic
factor, (Kumar et al. 2007b), which provides a fascinating link between positional
identity and proliferation.
Interestingly, the prod-1 promoter depicts two binding sites for meis gene products, which are also involved in PD determination during embryonic limb development, suggesting that RA induces similar mechanisms for axial determination during
limb development and regeneration (Shaikh et al. 2011).
Recently, microarray studies performed with RA-treated axolotl distal blastemas
after amputation, revealed gene transcriptional responses associated with RAinduced proximodistal duplications (Nguyen et al. 2017; Voss et al. 2018). At least
100 genes were up-regulated including RA signaling genes (cyp26a1, cyp26B1,
nrip1), several genes normally expressed in proximal developing limb buds in other
vertebrates (meis1, meis2, pbx1, arid5b, fibin„ epha7, fabp2, uroplakin3a, tcaf1,
keratin19, rnd3), and ECM genes (aggrecan and brevican). Conversely, other genes
that are normally expressed in the distal part of the developing limb bud were downregulated, including lhx9, zic5, lmo1, lhx2, spry1, msx2 and hoxa13. Again, the
similarities between genes involved in PD specification in the limb bud and the
limb regenerate are striking. Figure 4.4 shows the distribution of the RA enzymatic
machinery and the effect of an excess or a deficiency of RA on the regenerating
amphibian limb. A summary of the RA-dependent gene cascades is shown in Fig. 4.5.
Chick Limb Development
Is RA Required?
Disulphiram application at chick pre-limb bud or early limb bud stages, abolished
forelimb development as well as shh expression in the ZPA (Stratford et al. 1996).