108
M. Maden
buds show loss of digits and fused long bones (typical teratological phenotypes).
The girdles are intact (Yashiro et al. 2004).
The Effects of Excess RA
In mice, excess RA cannot be administered locally to the developing limb bud on
a bead. Therefore, RA is routinely administered by oral gavage of the mother at
particular days of gestation. Amazingly, when administered at blastocyst and pregastrulation stages (days 4.5–5.5), i.e. long before limb bud formation, RA has a
dramatic effect on caudal regions resulting in (1) pairs of symmetrical hindlimb buds,
(2) duplication of the lower body axis and tail with extra pairs of hindlimbs, pelvic
girdles, and tails, and (3) hindlimb twinning and polydactylous limbs (Rutledge et al.
1994; Niederreither et al. 1996; Liao and Collins 2008, Fig. 4.3d). The ectopic limb
buds show shh, fgf-4, fgf-8, msx1, pitx1, tbx-4 and hoxb-8 expression. Only very
rarely are the forelimbs affected. These effects are remarkably similar to those of RA
on zebrafish pectoral fin multiplication (Vandersea et al. 1998; Fig. 4.3a, Fig. 4.4)
and on amphibian tail regeneration where a mass of ectopic hindlimbs develop on
the end of a regenerating tail (Mohanti-Hejmadi et al. 1992; Maden 1993; Fig. 4.3c),
again emphasizing commonality of the effects of RA on limb development and
regeneration.
In complete contrast to these results, when RA is administered just prior to or
during limb development stages, RA has a negative effect—the limbs display fusion
of long bones, loss or shortening of long bones, and loss of digits, with the precise level
and severity of effect correlated with a typical stage specificity (Shenefelt 1972; Lee
et al. 2004). These effects are the classical effects of RA as a teratogen in mammals,
including humans.
Another method of increasing RA in the limb bud is to delete the gene for cyp26b1,
the enzyme that catabolizes RA. Lack of CYP26b1 leads to excess RA in the distal
region of the limb buds, which results in loss of digits and fused long bones (Yoshiro
et al. 2004). AP and DV patterning genes are normal in these limb buds, but the distal
hox genes d-12, d-13 and a-13 are reduced, and the normally proximal meis1 and
meis2 genes expand into distal regions. A RARE-lacZ reporter mouse confirmed the
presence of an expanded domain of RA in distal regions, and distal cells, labelled
with a lipophilic dye, were shown to move into the zeugopodial region rather than
remain in the autopod. Together, the data show a clear PD effect of excess RA on the
limb. Figure 4.4 summarizes the distribution of the RA enzymatic machinery and
the effect of an excess or a deficiency of RA on the mouse limb bud and a summary
of the RA-dependent gene cascades is shown in Fig. 4.5.
Recapitulation of RA Effects on Limb Development Across Species
A summary of the RA-dependent gene cascades described above is shown in Fig. 4.5
where the outline of the limb is drawn along with the adjacent somites. In all cases
M. Maden
buds show loss of digits and fused long bones (typical teratological phenotypes).
The girdles are intact (Yashiro et al. 2004).
The Effects of Excess RA
In mice, excess RA cannot be administered locally to the developing limb bud on
a bead. Therefore, RA is routinely administered by oral gavage of the mother at
particular days of gestation. Amazingly, when administered at blastocyst and pregastrulation stages (days 4.5–5.5), i.e. long before limb bud formation, RA has a
dramatic effect on caudal regions resulting in (1) pairs of symmetrical hindlimb buds,
(2) duplication of the lower body axis and tail with extra pairs of hindlimbs, pelvic
girdles, and tails, and (3) hindlimb twinning and polydactylous limbs (Rutledge et al.
1994; Niederreither et al. 1996; Liao and Collins 2008, Fig. 4.3d). The ectopic limb
buds show shh, fgf-4, fgf-8, msx1, pitx1, tbx-4 and hoxb-8 expression. Only very
rarely are the forelimbs affected. These effects are remarkably similar to those of RA
on zebrafish pectoral fin multiplication (Vandersea et al. 1998; Fig. 4.3a, Fig. 4.4)
and on amphibian tail regeneration where a mass of ectopic hindlimbs develop on
the end of a regenerating tail (Mohanti-Hejmadi et al. 1992; Maden 1993; Fig. 4.3c),
again emphasizing commonality of the effects of RA on limb development and
regeneration.
In complete contrast to these results, when RA is administered just prior to or
during limb development stages, RA has a negative effect—the limbs display fusion
of long bones, loss or shortening of long bones, and loss of digits, with the precise level
and severity of effect correlated with a typical stage specificity (Shenefelt 1972; Lee
et al. 2004). These effects are the classical effects of RA as a teratogen in mammals,
including humans.
Another method of increasing RA in the limb bud is to delete the gene for cyp26b1,
the enzyme that catabolizes RA. Lack of CYP26b1 leads to excess RA in the distal
region of the limb buds, which results in loss of digits and fused long bones (Yoshiro
et al. 2004). AP and DV patterning genes are normal in these limb buds, but the distal
hox genes d-12, d-13 and a-13 are reduced, and the normally proximal meis1 and
meis2 genes expand into distal regions. A RARE-lacZ reporter mouse confirmed the
presence of an expanded domain of RA in distal regions, and distal cells, labelled
with a lipophilic dye, were shown to move into the zeugopodial region rather than
remain in the autopod. Together, the data show a clear PD effect of excess RA on the
limb. Figure 4.4 summarizes the distribution of the RA enzymatic machinery and
the effect of an excess or a deficiency of RA on the mouse limb bud and a summary
of the RA-dependent gene cascades is shown in Fig. 4.5.
Recapitulation of RA Effects on Limb Development Across Species
A summary of the RA-dependent gene cascades described above is shown in Fig. 4.5
where the outline of the limb is drawn along with the adjacent somites. In all cases