104
M. Maden
The Effects of Excess RA
Application of excess RA has teratogenic effects on chick limb development. When
RA in a DMSO solution is simply dropped onto limb buds, the resulting limbs lack
digits or have abnormally long bones (Larsen and Janners 1987).
However, if applied by implanting beads or other absorptive materials at prelimb stages or to the anterior margin of the limb bud, RA has a striking effect on
anteroposterior axis formation resulting in digit duplications (Fig. 4.1c), which are
position and concentration dependent (Tickle et al. 1982, 1985; Summerbell 1983).
The radius and ulna of the forearm are also frequently duplicated, usually with
2 ulnas and a missing radius. A complete limb with a duplicated humerus is never
seen. Similar effects are seen in the hindlimb (Wilde et al. 1987). Even after applying
RA to the embryonic flank at stages long before the limb bud appears, the limbs still
show AP duplications, primarily in the digits. If the dose of RA is too high, then
defective limbs are produced, with a reduced number of digits, or the limb can be
completely missing, except for the scapula.
RA has also been shown to affect the PD axis (Tamura et al. 1997). Indeed, when
the tissue adjacent to a RA bead placed on the anterior margin of a chick limb bud was
grafted to different PD segments of an earlier stage, the graft produced a complete
limb with humerus, radius-ulna, and digits. Proximalization was also seen when
chick RA-treated tissue grafts were placed into a quail limb bud—the grafted chick
cells could be seen populating the entire PD length of the quail limb rather than just
the digital levels which would have normally formed without RA treatment.
Several studies analyzed the genes that are induced or repressed by RA during
posteriorization of the chick limb bud (Izpisua-Belmonte et al. 1991; Nohno et al.
1991; Riddle et al. 1993; Helms et al. 1996). A recent microarray study revealed
large changes in gene expression levels, with 292 genes increased and 996 decreased
(Pickering et al. 2017). Application of RA-soaked beads to the anterior margin of
wing buds increased the expression of several patterning genes that are normally
expressed in the posterior part of the limb bud (hoxd-11, hoxd-12, hoxd-13, fgf-4,
bmp-2, dHAND and shh) and repressed genes that are normally expressed in the
anterior limb bud (bmp-4, lhx9, msx2, alx4 and gli3).
Interestingly, the earliest genes induced by ectopic RA (and not by a ZPA graft)
are RARβ, and hoxb-8 (Lu et al. 1997b; Stratford et al. 1997). It has been suggested
that hoxb-8 would be a primary target of RA. Importantly, hoxb-8 is the only gene
showing a difference between fore and hindlimb buds. It is rapidly induced by RA in
the forelimb, but not induced nor ever expressed in the hindlimb bud (Stratford et al.
1997). In contrast, shh would not be a direct target of RA and would be regulated
by fgf-4 through the AER (Niswander et al. 1994). Hoxd gene induction and gli3
repression would cooperate to suppress an anterior development program and induce
a posterior program.
The genes involved in proximalization have been also analyzed and were identified
as meis1 and meis2 (Mercarder et al. 2000). These genes are strikingly universal
across chick and mouse limb buds and in axolotl limb regeneration. Meis1 and
meis2 are expressed shortly after the emergence of the limb bud in the lateral plate
M. Maden
The Effects of Excess RA
Application of excess RA has teratogenic effects on chick limb development. When
RA in a DMSO solution is simply dropped onto limb buds, the resulting limbs lack
digits or have abnormally long bones (Larsen and Janners 1987).
However, if applied by implanting beads or other absorptive materials at prelimb stages or to the anterior margin of the limb bud, RA has a striking effect on
anteroposterior axis formation resulting in digit duplications (Fig. 4.1c), which are
position and concentration dependent (Tickle et al. 1982, 1985; Summerbell 1983).
The radius and ulna of the forearm are also frequently duplicated, usually with
2 ulnas and a missing radius. A complete limb with a duplicated humerus is never
seen. Similar effects are seen in the hindlimb (Wilde et al. 1987). Even after applying
RA to the embryonic flank at stages long before the limb bud appears, the limbs still
show AP duplications, primarily in the digits. If the dose of RA is too high, then
defective limbs are produced, with a reduced number of digits, or the limb can be
completely missing, except for the scapula.
RA has also been shown to affect the PD axis (Tamura et al. 1997). Indeed, when
the tissue adjacent to a RA bead placed on the anterior margin of a chick limb bud was
grafted to different PD segments of an earlier stage, the graft produced a complete
limb with humerus, radius-ulna, and digits. Proximalization was also seen when
chick RA-treated tissue grafts were placed into a quail limb bud—the grafted chick
cells could be seen populating the entire PD length of the quail limb rather than just
the digital levels which would have normally formed without RA treatment.
Several studies analyzed the genes that are induced or repressed by RA during
posteriorization of the chick limb bud (Izpisua-Belmonte et al. 1991; Nohno et al.
1991; Riddle et al. 1993; Helms et al. 1996). A recent microarray study revealed
large changes in gene expression levels, with 292 genes increased and 996 decreased
(Pickering et al. 2017). Application of RA-soaked beads to the anterior margin of
wing buds increased the expression of several patterning genes that are normally
expressed in the posterior part of the limb bud (hoxd-11, hoxd-12, hoxd-13, fgf-4,
bmp-2, dHAND and shh) and repressed genes that are normally expressed in the
anterior limb bud (bmp-4, lhx9, msx2, alx4 and gli3).
Interestingly, the earliest genes induced by ectopic RA (and not by a ZPA graft)
are RARβ, and hoxb-8 (Lu et al. 1997b; Stratford et al. 1997). It has been suggested
that hoxb-8 would be a primary target of RA. Importantly, hoxb-8 is the only gene
showing a difference between fore and hindlimb buds. It is rapidly induced by RA in
the forelimb, but not induced nor ever expressed in the hindlimb bud (Stratford et al.
1997). In contrast, shh would not be a direct target of RA and would be regulated
by fgf-4 through the AER (Niswander et al. 1994). Hoxd gene induction and gli3
repression would cooperate to suppress an anterior development program and induce
a posterior program.
The genes involved in proximalization have been also analyzed and were identified
as meis1 and meis2 (Mercarder et al. 2000). These genes are strikingly universal
across chick and mouse limb buds and in axolotl limb regeneration. Meis1 and
meis2 are expressed shortly after the emergence of the limb bud in the lateral plate