102
M. Maden
Comparable experiments have not been done on the hindlimb. Treatment of the presumptive forelimb region with beads impregnated with both RAR and RXR antagonists produced a range of defects from complete agenesis of the limb to a misshapen
hand plate with a loss of structures in a posterior to anterior direction (Helms et al.
1996). When the antagonist-treated limb buds were rescued with RA grafted under
the apical ectodermal ridge (AER), some of the buds showed a reversal of AP polarity,
confirming the role of RA in the establishment of the ZPA.
The main genes that failed to appear in the mesoderm of the antagonist-treated
limb buds are hoxb-8 and shh (which are thought to be responsible for establishing
the forelimb ZPA), and bmp-2 (Lu et al. 1997a, b; Stratford et al. 1997). Surprisingly,
neither hoxd-11 nor hoxd-13 were affected (Lu et al. 1997a, b), despite the fact that
these genes are readily induced by ectopic RA (Stratford et al. 1997). In the apical
ectodermal ridge (AER), fgf-8 and bmp-2 were not affected. This result cautions
against making conclusions about genes that are controlled by RA during normal
development on the basis of those that can be induced by excess RA.
Interestingly, in RA-deficient quail embryos, which lack detectable retinoids and
eventually die of heart defects similar to raldh2
−/− mice, fore and hindlimb buds
appeared. However, they were much reduced in size, and genes representing different
aspects of limb polarity were affected (Stratford et al. 1999). As an example, concerning the anteroposterior axis, hoxb-8 was more widely expressed, while bmp-2 and
shh were down-regulated. Concerning the apical ectodermal genes, fgf-4 was down
regulated, whereas bmp-2 and fgf-8 were unaffected. Genes involved in dorsoventral
polarity were also affected—wnt-7a, normally confined to the dorsal ectoderm was
shifted to the ventral ectoderm, while the dorsal gene, lmx-1, was spread into the
ventral mesoderm. Consequently, En-1, normally expressed in the ventral ectoderm
was absent. These results show that RA plays a role not only in the organization of
the PD and AP axis, but also in the organization of the dorsoventral axis.
Is RA Present?
Discovery of the effects of RA on duplication of the AP axis of the chick limb bud
inspired a body of work centered on understanding how RA influenced development
of the AP axis. First, HPLC analysis confirmed the presence of all-trans RA (tRA)
in whole limb buds (Thaller and Eichele 1987), and indicated that RA was present at
similar levels in the posterior part of the limb bud (consisting of about one quarter of
the bud) and in the anterior part (consisting of the remaining three quarters). However,
when normalized to DNA content, the posterior domain had 2.6 times more RA
than the anterior domain. The concentration in the posterior part was approximately
50 nM.
RA was also detected in the chick wing bud by placing tissues on a lawn of F9
reporter cells expressing the RARE-lacZ gene and quantifying the numbers of blue
cells induced: a two-fold higher level of RA was detected in posterior halves of wing
buds compared to anterior halves (Sonneveld et al. 1999). Analysis of the RARβ2
transcripts also indicated that this RAR is enriched three-fold in the posterior limb
M. Maden
Comparable experiments have not been done on the hindlimb. Treatment of the presumptive forelimb region with beads impregnated with both RAR and RXR antagonists produced a range of defects from complete agenesis of the limb to a misshapen
hand plate with a loss of structures in a posterior to anterior direction (Helms et al.
1996). When the antagonist-treated limb buds were rescued with RA grafted under
the apical ectodermal ridge (AER), some of the buds showed a reversal of AP polarity,
confirming the role of RA in the establishment of the ZPA.
The main genes that failed to appear in the mesoderm of the antagonist-treated
limb buds are hoxb-8 and shh (which are thought to be responsible for establishing
the forelimb ZPA), and bmp-2 (Lu et al. 1997a, b; Stratford et al. 1997). Surprisingly,
neither hoxd-11 nor hoxd-13 were affected (Lu et al. 1997a, b), despite the fact that
these genes are readily induced by ectopic RA (Stratford et al. 1997). In the apical
ectodermal ridge (AER), fgf-8 and bmp-2 were not affected. This result cautions
against making conclusions about genes that are controlled by RA during normal
development on the basis of those that can be induced by excess RA.
Interestingly, in RA-deficient quail embryos, which lack detectable retinoids and
eventually die of heart defects similar to raldh2
−/− mice, fore and hindlimb buds
appeared. However, they were much reduced in size, and genes representing different
aspects of limb polarity were affected (Stratford et al. 1999). As an example, concerning the anteroposterior axis, hoxb-8 was more widely expressed, while bmp-2 and
shh were down-regulated. Concerning the apical ectodermal genes, fgf-4 was down
regulated, whereas bmp-2 and fgf-8 were unaffected. Genes involved in dorsoventral
polarity were also affected—wnt-7a, normally confined to the dorsal ectoderm was
shifted to the ventral ectoderm, while the dorsal gene, lmx-1, was spread into the
ventral mesoderm. Consequently, En-1, normally expressed in the ventral ectoderm
was absent. These results show that RA plays a role not only in the organization of
the PD and AP axis, but also in the organization of the dorsoventral axis.
Is RA Present?
Discovery of the effects of RA on duplication of the AP axis of the chick limb bud
inspired a body of work centered on understanding how RA influenced development
of the AP axis. First, HPLC analysis confirmed the presence of all-trans RA (tRA)
in whole limb buds (Thaller and Eichele 1987), and indicated that RA was present at
similar levels in the posterior part of the limb bud (consisting of about one quarter of
the bud) and in the anterior part (consisting of the remaining three quarters). However,
when normalized to DNA content, the posterior domain had 2.6 times more RA
than the anterior domain. The concentration in the posterior part was approximately
50 nM.
RA was also detected in the chick wing bud by placing tissues on a lawn of F9
reporter cells expressing the RARE-lacZ gene and quantifying the numbers of blue
cells induced: a two-fold higher level of RA was detected in posterior halves of wing
buds compared to anterior halves (Sonneveld et al. 1999). Analysis of the RARβ2
transcripts also indicated that this RAR is enriched three-fold in the posterior limb