4 RA Signaling in Limb Development and Regeneration …
89
The concept of a morphogen gradient released from a source and acting to
induce differentiation according to a concentration dependent mechanism became the
bedrock of developmental biology because it explained so much of the long history
of experimental embryology which began in the 1860s. The search was on, therefore,
for molecular evidence in favor of this theory and, of course, for the identification of
morphogens themselves.
When the remarkable effects of RA on the developing chick limb bud and the
regenerating axolotl limb were described at precisely the same time (Tickle et al.
1982; Maden 1982), the findings were seen as very dramatic because this single
molecule displayed all of the properties of a long-sought after morphogen in the
limb or, for that matter, anywhere else in the embryo.
History
Saunders and Gasseling (1968) and Tickle et al. (1975) identified a region of the chick
limb bud with the properties of an organizer, which they called the zone of polarizing
activity (ZPA, Fig. 4.1a) When a ZPA was transplanted from the posterior margin
of the limb bud to the opposite side of another limb bud, a six-digit limb (instead
of the normal 3 digit chick wing) was produced, with the digits arranged in mirrorimage to each other (Fig. 4.1b, c). This anomalous behavior after grafting could be
explained in terms of Wolpert’s positional information by proposing that the ZPA
released a morphogen that diffuses across the limb bud from the posterior side to the
anterior side to generate a concentration gradient, such that different concentrations
of the morphogen induce formation of different digits—high concentrations induce
digit 4, intermediate concentrations induce digit 3, low concentrations induce digit
2 (there are only 3 digits in the chicken wing). Then the question was whether
the ZPA released a morphogen for limb bud development. In favor of this concept,
extracellular gradients operate over a field size of fewer than 100 cells and a maximum
distance of 1 mm for a low molecular weight morphogen (Crick 1970), which fits
well with a limb bud whose volume is approximately 0.009 mm
3 .
However, concepts devised to explain regenerating limb phenomena were very
different. First, there is a huge size difference between the developing limb bud
and the regenerating blastema. Indeed, regenerating blastemas on large axolotls can
be 2 mm or more in each dimension, with volumes 1000 times greater than those
of developing limb buds. Thus, instead of a morphogen gradient model, prevailing
evidence supported a polar co-ordinate model (French et al. 1976) where local cell
surface interactions between two adjacent cells (and not a distant source of morphogen) direct regeneration. As an example, when a regeneration blastema is cut
off from the limb, rotated 180° and stuck back onto the same stump, frequently the
blastema de-rotates and ends up in exactly the same position where it started (Maden
1978). As another example, when a wrist blastema is cut off from the limb, moved
proximally to the upper arm level, and the limb then amputated through the upper
arm level, the grafted blastema moves distally. Then as the limb regenerates, it ceases
89
The concept of a morphogen gradient released from a source and acting to
induce differentiation according to a concentration dependent mechanism became the
bedrock of developmental biology because it explained so much of the long history
of experimental embryology which began in the 1860s. The search was on, therefore,
for molecular evidence in favor of this theory and, of course, for the identification of
morphogens themselves.
When the remarkable effects of RA on the developing chick limb bud and the
regenerating axolotl limb were described at precisely the same time (Tickle et al.
1982; Maden 1982), the findings were seen as very dramatic because this single
molecule displayed all of the properties of a long-sought after morphogen in the
limb or, for that matter, anywhere else in the embryo.
History
Saunders and Gasseling (1968) and Tickle et al. (1975) identified a region of the chick
limb bud with the properties of an organizer, which they called the zone of polarizing
activity (ZPA, Fig. 4.1a) When a ZPA was transplanted from the posterior margin
of the limb bud to the opposite side of another limb bud, a six-digit limb (instead
of the normal 3 digit chick wing) was produced, with the digits arranged in mirrorimage to each other (Fig. 4.1b, c). This anomalous behavior after grafting could be
explained in terms of Wolpert’s positional information by proposing that the ZPA
released a morphogen that diffuses across the limb bud from the posterior side to the
anterior side to generate a concentration gradient, such that different concentrations
of the morphogen induce formation of different digits—high concentrations induce
digit 4, intermediate concentrations induce digit 3, low concentrations induce digit
2 (there are only 3 digits in the chicken wing). Then the question was whether
the ZPA released a morphogen for limb bud development. In favor of this concept,
extracellular gradients operate over a field size of fewer than 100 cells and a maximum
distance of 1 mm for a low molecular weight morphogen (Crick 1970), which fits
well with a limb bud whose volume is approximately 0.009 mm
3 .
However, concepts devised to explain regenerating limb phenomena were very
different. First, there is a huge size difference between the developing limb bud
and the regenerating blastema. Indeed, regenerating blastemas on large axolotls can
be 2 mm or more in each dimension, with volumes 1000 times greater than those
of developing limb buds. Thus, instead of a morphogen gradient model, prevailing
evidence supported a polar co-ordinate model (French et al. 1976) where local cell
surface interactions between two adjacent cells (and not a distant source of morphogen) direct regeneration. As an example, when a regeneration blastema is cut
off from the limb, rotated 180° and stuck back onto the same stump, frequently the
blastema de-rotates and ends up in exactly the same position where it started (Maden
1978). As another example, when a wrist blastema is cut off from the limb, moved
proximally to the upper arm level, and the limb then amputated through the upper
arm level, the grafted blastema moves distally. Then as the limb regenerates, it ceases