4 RA Signaling in Limb Development and Regeneration …
107
Is RA Present?
RA has been visualized in RARE-lacZ reporter mice, and the types and levels of
retinoids in mouse limb buds have been quantified by HPLC. Day 10.5 and 13.5
limb buds contain about 30 nM of tRA (a concentration similar to that seen in the
chick limb bud). There are no consistent differences in tRA levels in the anterior and
posterior halves of the limb buds. The highest levels of tRA (4-fold higher than limb
buds) are seen in the neural tube. Retinol levels are about 5-fold higher than RA in
the limb buds. There are no didehydro retinoids in the mouse (Satre and Kochhar
1989; Horton and Maden 1995; Scott et al. 1994).
Results with the RAREhspLacZ reporter mouse showed that day 10.5 limb buds
were negative for the β-gal product even though HPLC analysis revealed 30 nM RA
in limb bud tissues and even though a strong β-gal signal was detected in the adjacent
neural tube, somites, and lateral plate from the beginning of the spinal cord to the tail
bud. In fact, β-gal expression was observed throughout the limb bud at day 9.5, but
was gone by day 9.75 in the distal third of the limb bud (Mic et al. 2004). Then, at
day 10.5, there was some β-gal staining in the proximal part of the limb bud adjacent
to the strong staining of the lateral plate, but most of the limb bud remained β-gal
negative (and by implication RA-free), until day 14.5, when there was β-gal staining
in the interdigital region of the fully differentiated limbs. These RARE-lacZ results
nicely reflect raldh2 expression domains in adjacent tissues and cyp26b1 domains
in the distal half of the limb bud. The reporter mouse experiments demonstrate that
RA diffuses into the limb bud and is eliminated distally.
Where Is RA Generated?
As in all other vertebrates, in the mouse, raldh2 expression begins after gastrulation
(day 7.0–7.5) in the lateral mesoderm, and then is present in the condensing somites
and adjacent lateral plate mesoderm (Zhao et al. 1996; Niederreither et al. 1999).
Its expression in somites intensifies after their formation, then declines, but there
is no raldh2 expression in the limb bud at these stages). However, during forelimb
bud extension (day 10.5–11.5), an additional site of raldh2 expression appears at
the proximal base of the limb bud, adjacent to the coelomic cavity, exactly as in the
chick embryo (Berggren et al. 1999). Thus, at initial stages, limb buds do not express
any RA enzymes, but at later differentiation stages they express raldh2 in a proximal
domain beside the flank, in a central domain around the arteries, and then in the
interdigital region as the digits form (Niederreither et al. 1999). Rdh10 is expressed
in the dorsal somites, intermediate mesoderm, lateral plate and proximal forelimb
bud mesenchyme of both fore and hindlimb buds at day 9.5. The most intense level
of expression is in the posterior ZPA.
The RA-free distal domain in the limb bud is due to the appearance of cyp26b1
expression in the distal ectoderm and sub-ectodermal zone (MacLean et al. 2001;
Yashiro et al. 2004). When this RA sink is deleted, then both fore- and hindlimb
107
Is RA Present?
RA has been visualized in RARE-lacZ reporter mice, and the types and levels of
retinoids in mouse limb buds have been quantified by HPLC. Day 10.5 and 13.5
limb buds contain about 30 nM of tRA (a concentration similar to that seen in the
chick limb bud). There are no consistent differences in tRA levels in the anterior and
posterior halves of the limb buds. The highest levels of tRA (4-fold higher than limb
buds) are seen in the neural tube. Retinol levels are about 5-fold higher than RA in
the limb buds. There are no didehydro retinoids in the mouse (Satre and Kochhar
1989; Horton and Maden 1995; Scott et al. 1994).
Results with the RAREhspLacZ reporter mouse showed that day 10.5 limb buds
were negative for the β-gal product even though HPLC analysis revealed 30 nM RA
in limb bud tissues and even though a strong β-gal signal was detected in the adjacent
neural tube, somites, and lateral plate from the beginning of the spinal cord to the tail
bud. In fact, β-gal expression was observed throughout the limb bud at day 9.5, but
was gone by day 9.75 in the distal third of the limb bud (Mic et al. 2004). Then, at
day 10.5, there was some β-gal staining in the proximal part of the limb bud adjacent
to the strong staining of the lateral plate, but most of the limb bud remained β-gal
negative (and by implication RA-free), until day 14.5, when there was β-gal staining
in the interdigital region of the fully differentiated limbs. These RARE-lacZ results
nicely reflect raldh2 expression domains in adjacent tissues and cyp26b1 domains
in the distal half of the limb bud. The reporter mouse experiments demonstrate that
RA diffuses into the limb bud and is eliminated distally.
Where Is RA Generated?
As in all other vertebrates, in the mouse, raldh2 expression begins after gastrulation
(day 7.0–7.5) in the lateral mesoderm, and then is present in the condensing somites
and adjacent lateral plate mesoderm (Zhao et al. 1996; Niederreither et al. 1999).
Its expression in somites intensifies after their formation, then declines, but there
is no raldh2 expression in the limb bud at these stages). However, during forelimb
bud extension (day 10.5–11.5), an additional site of raldh2 expression appears at
the proximal base of the limb bud, adjacent to the coelomic cavity, exactly as in the
chick embryo (Berggren et al. 1999). Thus, at initial stages, limb buds do not express
any RA enzymes, but at later differentiation stages they express raldh2 in a proximal
domain beside the flank, in a central domain around the arteries, and then in the
interdigital region as the digits form (Niederreither et al. 1999). Rdh10 is expressed
in the dorsal somites, intermediate mesoderm, lateral plate and proximal forelimb
bud mesenchyme of both fore and hindlimb buds at day 9.5. The most intense level
of expression is in the posterior ZPA.
The RA-free distal domain in the limb bud is due to the appearance of cyp26b1
expression in the distal ectoderm and sub-ectodermal zone (MacLean et al. 2001;
Yashiro et al. 2004). When this RA sink is deleted, then both fore- and hindlimb