Activation of a single retinoic acid receptor isoform mediates proximodistal respecification.

Abstract

The regenerating limb of urodele amphibians is an important system for evaluating the effects of retinoic acid (RA) on pattern formation. Regeneration proceeds by local formation of the blastema, a mesenchymal growth zone which normally only gives rise to structures distal to its level of origin. RA can respecify proximodistal identity in amphibian limb regeneration, and this activity of RA on the blastema is observed in two contexts. First, exposure to RA proximalizes a distal blastema resulting in duplication of structures proximal to the level of amputation. Second, after transplantation of a distal blastema to a proximal stump, the transplanted cells normally make only a minor contribution to the intercalary regenerate, but if transplanted cells are exposed to RA they occupy positions proximal to their level of origin and contribute to the regeneration of the intermediate tissue. Multiple isoforms of RA receptors (RARs) are expressed in the newt limb and are thought to mediate the respecification of positional identity.
To identify which receptor(s) mediates proximodistal respecification, we have used the biolistics (particle bombardment) technique to transfect the blastemal mesenchyme with plasmids encoding chimeric proteins containing partial amino-acid sequences of the various newt RAR isoforms fused to a partial sequence of the thyroid hormone (3,5, 3'-triiodothyronine; T3) receptor. We then used T3 treatment to selectively activate individual RAR isoforms in vivo. By analyzing the distributions of transfected cells in regenerates derived from distal-to-proximal transplantation we find that activation of a single RAR isoform, delta 2, specifically mediates proximalization.
These results demonstrate that the ability of RA to respecify proximodistal identity is mediated by a specific RAR isoform, delta 2. Activation of the RA pathway in individual cells indicates that positional respecification can be cell-autonomous. RA can respecify axial identity in several contexts in vertebrate development, but this is the first case where the RAR mediating respecification has been identified.

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Activation of a single retinoic acid receptor isoform mediates
proximodistal respecification
Lauren T. Pecorino, Alan Entwistle and Jeremy P. Brockes
Background: The regenerating limb of urodele amphibians is an important
system for evaluating the effects of retinoic acid (RA) on pattern formation.
Regeneration proceeds by local formation of the blastema, a mesenchymal
growth zone which normally only gives rise to structures distal to its level of
origin. RA can respecify proximodistal identity in amphibian limb regeneration,
and this activity of RA on the blastema is observed in two contexts. First,
exposure to RA proximalizes a distal blastema resulting in duplication of
structures proximal to the level of amputation. Second, after transplantation of a
distal blastema to a proximal stump, the transplanted cells normally make only a
minor contribution to the intercalary regenerate, but if transplanted cells are
exposed to RA they occupy positions proximal to their level of origin and
contribute to the regeneration of the intermediate tissue. Multiple isoforms of RA
receptors (RARs) are expressed in the newt limb and are thought to mediate the
respecification of positional identity.
Results: To identify which receptor(s) mediates proximodistal respecification, we
have used the biolistics (particle bombardment) technique to transfect the
blastemal mesenchyme with plasmids encoding chimeric proteins containing
partial amino-acid sequences of the various newt RAR isoforms fused to a partial
sequence of the thyroid hormone (3,5,3′-triiodothyronine; T3) receptor. We then
used T3treatment to selectively activate individual RAR isoforms in vivo. By
analyzing the distributions of transfected cells in regenerates derived from distal-
to-proximal transplantation we find that activation of a single RAR isoform, ?2,
specifically mediates proximalization.
Conclusions: These results demonstrate that the ability of RA to respecify
proximodistal identity is mediated by a specific RAR isoform, ?2. Activation of the
RA pathway in individual cells indicates that positional respecification can be
cell-autonomous. RA can respecify axial identity in several contexts in vertebrate
development, but this is the first case where the RAR mediating respecification
has been identified.
Background
Our current understanding of pattern formation in devel-
opment and regeneration is that cells possess positional
identity which specifies their location on a developmental
axis. It remains unclear how positional identity is encoded
at the molecular level, particularly in relation to those cel-
lular properties, such as proliferation, adhesion or migra-
tion, that might be important in tissue patterning. The
ability of retinoic acid (RA) to respecify axial identity in
different contexts offers an important experimental
approach to this question, and one prominent example is
its effect on the proximodistal axis during limb regenera-
tion in urodele amphibians, the only vertebrates able to
regenerate their limbs as adults.
Regeneration in urodeles, such as the newt, proceeds by
local formation of a blastema [1,2], a zone of mesenchymal
progenitor cells which normally only gives rise to struc-
tures distal to its level of origin. Thus, a wrist blastema
forms a hand, whereas a shoulder blastema regenerates an
entire arm. If a distal (wrist) blastema is grafted to a proxi-
mal (shoulder) stump, a normal limb is regenerated in
which the hand is derived from the blastema, but the
intermediate structures between the shoulder and wrist
are derived from the stump by the process of intercalation
[3]. Thus, RA can proximalize a regenerate in two con-
texts: distal amputation and transplantation of a distal
blastema. In the first, an RA-treated blastema gives rise to
structures proximal to the level of amputation, resulting in
a partial duplication of the proximodistal axis [4–6]. In the
second, the contribution of cells to the intercalary regener-
ate is increased if a distal blastema is exposed to RA prior
to grafting to a proximal stump. RA-treated transplanted
cells are detected in locations proximal to their level of
Address: The Ludwig Institute for Cancer
Research and Department of Biochemistry and
Molecular Biology, University College London,
91 Riding House Street, London W1P 8BT, UK.
Correspondence to: Jeremy P. Brockes
E-mail address: jerbro@ludwig.ucl.ac.uk
Received: 13 February 1996
Accepted: 6 March 1996
Current Biology 1996, Vol 6 No 5:563–569
© Current Biology Ltd ISSN 0960-9822
Research Paper563

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origin and contribute to formation of the intermediate
structures [7,8]. Thus, proximodistal intercalation provides
a context to assay positional identity at a cellular level.
The proximodistal respecification of the blastema is
thought to be mediated by activation of one or more
members of the RA receptor (RAR) family of nuclear
receptors. It is noteworthy that the synthetic retinoid
agonist TTNPB (ethyl p-[(E)-2-(tetrahydro-tetramethyl-2-
naphthyl)-1-propenyl] benzoate) is potent at respecifying a
distal blastema [9], and is selective for the RARs, as com-
pared to RXRs, which are a distinct retinoid receptor
family [10]. The RXRs are presumably required in their
role as heterodimerization partners for the RARs, but it is
the latter that must be activated by ligand to induce proxi-
malization. The five newt RAR isoforms that have been
identified by cDNA cloning are expressed in both the
mesenchymal blastema and the epidermis [11–13]. We
have investigated whether selective activation of RARs in
mesenchymal cells leads to proximalization. In order to
activate a single isoform in newt limb cells, plasmids
encoding chimeric proteins containing the amino-acid
sequences of the RAR isoforms fused to the sequence
encoding the thyroid hormone (3,5,3′-triiodothyronine; T3)
receptor have been constructed. In these fusion proteins,
the ligand-binding domain (EF regions) of each RAR was
replaced with the corresponding region of the Xe nopus T3
receptor-? [14,15]. These receptors retain the DNA-
binding specificity of RARs, but activate transcription in
response to T3rather than RA. They can be distinguished
functionally in that only ??1, the chimera of RAR?1,
induced T3-dependent inhibition of cell proliferation in
culture [14,15]. In contrast, ??1b, but not ??1, was able to
mediate T3-dependent induction of an RA-responsive
antigen in the wound epidermis [16]. In this account we
have transfected the mesenchyme of distal limb blastemas
and used T3to activate the chimeric receptors in vivo. By
analyzing the distributions of transfected cells in inter-
calary regenerates we show that activation of a single
receptor, ?2, specifically mediates proximalization.
Results
The RA–T3 chimeric receptors (??1, ??2, ??1a, ??1b, ??2)
used in this study contain the DNA-binding domain of the
various RARs in the amino-terminal half and the ligand-
binding domain of the T3receptor in the carboxy-terminal
half of the molecule (Fig. 1). To investigate the role of
individual isoforms in proximodistal respecification, plas-
mids expressing a chimeric receptor and an alkaline phos-
phatase marker gene were introduced into mesenchymal
cells of a distal hindlimb newt blastema by biolistic trans-
fection (particle bombardment) [8,17,18]. The transfected
blastemas were grafted back to a proximal stump, and sub-
sequently exposed to dimethylsulfoxide (DMSO) vehicle
or to T3in DMSO (Fig. 2a). An example of such a regener-
ate with an intercalated region is shown in Figure 2b. Cells
expressing alkaline phosphatase in the regenerate were
detected in sections by enzyme-labelled fluorescence in
conjunction with laser scanning microscopy, and identified
semiautomatically using cell-counting
described previously [8]. The gold particles that were pro-
pelled into the blastemal cells by biolistic transfection co-
localized with fluorescent cells under high power
magnification (Fig. 2c) and this provided an additional
control for the specificity of detection (see [8]). The distri-
bution of transfected cells was analysed by dividing the
proximodistal axis into five defined regions (tip, 2, 3, 4,
base), as described previously [8]. The most distal region
(tip) lies within the grafted distal blastema. Regions 2, 3
and 4 span the intercalary regenerate, whereas the most
proximal region (base) is located within the stump. A
distribution of marked cells that peaks in the proximal
regions would be expected if cells are respecified upon
activation of the appropriate chimeric receptors.
software, as
When distal blastemal cells were transfected with a
mixture of all five chimeric receptors and subsequently
activated with T3, they showed a distribution that peaked
in the base; only 30 % of transfected cells occupied the
two most distal regions (Figs 3a, 4b). In contrast the limbs
exposed to DMSO had more than 50 % of the transfected
cells in the tip and in region 2 (Figs 3b, 4a). This distal
distribution was also seen when cells were transfected
with an alkaline phosphatase marker gene alone, and sub-
sequently exposed to DMSO or to T3(data not shown).
The proximal distribution therefore required both the
complement of chimeric receptors and activation with T3.
564 Current Biology 1996, Vol 6 No 5
Figure 1
Schematic representation of the five chimeric (?) receptors used in this
study. These receptors consist of the amino-terminal regions (A–D) of
the newt (Notopthalmus viridescens) RA receptors (red or grey) and
the carboxy-terminal regions, including the ligand-binding domain, of
the Xenopus T3receptor-? (blue). The DNA-binding site of the RARs is
within domain C. The two ?? receptors differ in their A regions as a
result of alternative splicing, as do ??1b and ??2, as indicated. The
??1a receptor differs from ??1b in that it contains an amino-terminal
extension, as indicated [13].
A
χα1
χα2
χδ1
χδ2
BCDEF
DNA-bindingLigand-binding
ab

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In order to investigate if activation of a subset of receptors
in distal cells could induce the proximal distribution, the
receptors were divided into two groups — the ??s (??1
and ??2) and the ??s (??1a, ??1b and ??2) — which were
tested separately. Transfection with the ?? group gave a
distal distribution (Fig. 4c). In contrast, after transfection
with the ?? group, less than 30 % of the marked cells were
found in the two most distal regions (Fig. 4d), a distribu-
tion comparable to that seen when plasmids encoding all
five chimeric receptors were transfected (compare Figs
4a,c). The ?? and ?? distributions were significantly differ-
ent (see legend to Fig. 4). In order to resolve the role of
specific ? isoforms, newts were transfected either with the
two ??1 isoforms, or with ??2 alone. These experiments
showed that the ??1s gave a distal distribution (Figs 4e,
5a), whereas ??2 gave a proximal distribution (Figs 4f, 5b);
the two distributions were significantly different (see
legend to Fig. 4). These results indicate that RAR?2 medi-
ates the ability of RA to proximalize the blastema, although
the decrease in the proportion of cells in the two most
proximal regions between all of the ?? isoforms and ??2
alone leaves open the possibility of some synergistic activ-
ity from the ?1-containing chimeras, but the latter had no
activity when transfected without ??2.
Discussion
We have used transfection with chimeric receptors fol-
lowed by treatment with T3 to selectively activate the RA
response pathway in the blastemal mesenchyme. It should
be noted that T3normally has no effect on pattern forma-
tion in limb regeneration, as shown in this and earlier
studies [19]. Two distinct but overlapping distributions
were seen after analysis of intercalary regenerates. One
corresponded to distal cells, and the other corresponded to
Research Paper Proximalization by chimeric RARs Pecorino et al. 565
Figure 2
(a) Experimental design for analyzing the
functional role of activated chimeric receptors
in proximodistal respecification in a
regenerating newt limb. A hindlimb distal
blastema (ankle) was removed three weeks
after amputation. The mesenchymal blastemal
cells of the cut surface of the blastema were
transfected with chimeric receptor expression
plasmids by particle bombardment (vertical
arrow) in a tissue culture well. The blastema
with transfected cells (red circles) was grafted
onto a forelimb proximal stump (shoulder)
using surgical glue. Newts were injected with
T3in DMSO, or DMSO vehicle, 1 day and
10 days after grafting. After three weeks, the
distribution of transfected cells was analyzed.
The intercalated intermediate zone is enclosed
by the dotted line. Two distributions are
shown: distal blastemal cells expressing
chimeric receptors that have not been
activated by T3(that is, they are not
proximalized) occupy a distal distribution along
the proximodistal axis (P–D); distal blastemal
cells expressing chimeric receptors activated
by T3(proximalized) occupy a proximal
distribution. (b) A photomicrograph of a
regenerating newt limb arising from the graft of
an ankle blastema to a shoulder stump (see
(a)). The white arrows mark the boundaries of
the intercalated region and the stump (bottom)
or the graft (top). (c) Laser scanning
micrograph showing an alkaline phosphatase-
positive cell detected by enzyme labelled
fluorescence (red), and gold particles detected
by both scattered light and DIC optics (yellow;
see [8]). Nearly all phosphatase-positive cells
contain gold particles on high power (× 63
objective) inspection; however, not all gold
particles are associated with fluorescence. The
muscle fibers show some endogenous
fluorescence.
(b)
(a)
(c)
Analyze distribution?
along P–D axis
Transfect?
χ receptors
Graft
Forelimb?
proximal?
stump
Hindlimb distal?
blastema
DMSO
T3
BaseTip
Base Tip
PD
PD

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cells proximalized after receptor activation. These distrib-
utions were comparable to those previously seen in control
and RA-treated limbs, respectively [8]. Because at most
one cell in 100 is transfected by biolistics, it is noteworthy
that activation of the appropriate isoform led to respecifi-
cation of cells surrounded by many untransfected neigh-
bours. We favour the interpretation that RA acts directly
on distal cells to change their proximodistal identity in a
cell-autonomous manner, although it is unclear if the iden-
tity of neighbours might change as a result of some local
secreted signal from the transfected cells. In this sense the
results are consistent with the recent demonstration that
embryonic Xe nopus cells respond directly to an activin
gradient [20], but are in contrast to the mechanism by
which RA is thought to respecify the anteroposterior axis in
the chick limb bud. Local application to the anterior margin
of the bud leads to induction of Sonic hedgehog [21], which
in turn acts on the apical ectodermal ridge to release other
signals that act on the mesenchyme to ultimately induce
digit duplication [22,23]. Our results make it unlikely that
epithelial–mesenchymal interactions are involved in
proximodistal respecification during regeneration.
Although the cellular basis of proximalization is not under-
stood, the change in axial location in the regenerate could
reflect differences in expression of surface markers that
affect adhesion or migration. There is indirect evidence for
adhesive differences between limb blastemas of different
566 Current Biology 1996, Vol 6 No 5
Figure 3
Confocal micrographs of longitudinal sections
of regenerating limbs transfected with all five
chimeric receptors and treated with either (a)
T3or (b) DMSO vehicle. Cells expressing
alkaline phosphatase were visualized using
enzyme-labelled fluorescence, as described
[8], and are shown in yellow. The distal tip is
oriented to the left, and the base of the limb to
the right. Note that activation of the chimeric
receptors with T3results in a proximal
distribution relative to the DMSO vehicle
control. Scale bar= 275 ?m.

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proximodistal levels [24,25], and it has recently been noted
that RA has stage-specific effects on neural crest migration
into particular branchial arches [26]. The change in proxi-
modistal identity is probably not linked to the RA-induced
growth arrest of blastemal cells, because this is mediated
in culture by the ?1 receptor [14,15], although this is not
yet established in vivo. Whether RA is an endogenous
signal in the regenerating limb remains to be determined,
although data on its distribution, metabolism and release
in this context are accumulating [27–29].
The interspecies conservation of the receptor genes sug-
gests that specific isoforms might mediate distinct aspects
of the response to RA [30]. Together with other data
showing that the chimeric newt receptors are associated
with different activities [14–16], the present results support
this hypothesis. The difference in activity between ?1 and
?2 is presumably a consequence of the ability of the ?2 A
region to recruit specific factors to the transcription
complex on certain RA-responsive promoters (see [15]).
There is considerable evidence from mouse mutants for
some degree of redundancy in RAR function during devel-
opment (see [31] for review), although it is noteworthy that
certain RA-induced malformations are apparently mediated
by specific RARs or RXRs [32–34], and that anterior trans-
formations of the axial skeleton are seen in some RAR?-
null mice [32]. The newt RAR?1 was originally named
because its A region is markedly divergent from other
vertebrate RARs but the A region of ?2 is 58 % identical at
the amino-acid level to mouse RAR?2 [11,13], hence ?2 is
probably the newt homolog of ?2. The identification of a
single RAR that can be activated in the mesenchyme to
give proximodistal respecification will facilitate efforts to
identify downstream targets of RA, and this should yield
new insights into the basis of positional identity.
Conclusions
In this report we have analyzed the ability of different
RARs to respecify the positional identity of blastemal cells
during limb regeneration. The data indicate that selective
activation of a specific RAR isoform, ?2, within a blastemal
cell can mediate this effect. These results suggest that
activation of specific RARs could play an important part in
axis formation during vertebrate morphogenesis.
Materials and methods
Animals
Newts were supplied by Charles D. Sullivan Co. (Nashville, Ten-
nessee). All surgical procedures were performed on newts anes-
thetized in 0.1 % Tricaine (Sigma). Hindlimb amputations and
transplantation of distal mid-bud stage blastemas to proximal stumps
were performed as previously described [8]. T3(approximately 0.5 mg
per 100 g body weight) or DMSO vehicle alone were administered by
intraperitoneal injection 1 day and 10 days after grafting.
Biolistic transfection
Distal mid-stage blastemas were transfected by biolistics using a parti-
cle delivery system (PDS-1000/He, Bio-Rad) with chimeric receptor
expression plasmids containing an alkaline phosphatase marker gene
[8,14–16] as described previously [8]. In brief, the blastema was
Research Paper Proximalization by chimeric RARs Pecorino et al. 567
Figure 4
Distributions of cells transfected with RA–T3
chimeric receptors in regenerating limbs.
Graphs show the sum of cells (X) from a
group of animals (N), as a function of position
along the proximodistal axis. Regenerates
were induced by transplanting a distal
blastema transfected with plasmids
expressing all five of the chimeric receptors
shown in Fig. 1 (a, b), or plasmids expressing
the ?? isoforms (c), the ?? isoforms (d), the
??1 isoforms (e), or ??2 alone (f). The
regenerates were then treated with DMSO
vehicle alone (a) or activated with T3(b–f). X
is 176, 673, 502, 230, 244, 325 and N is 3,
7, 7, 4, 5, 6 for a, b, c, d, e, and f, respectively.
A weighted analysis of this data using the root
mean square value to ensure that an individual
animal with a relatively high or low number of
cells does not make a disproportionate
contribution (see [8]) gave near-identical
distributions. Using the chi squared test, the
distributions of a and b, c and d, and e and f
are significantly different pairs (P < 0.01).
70?
35?
0
200?
100?
0
100?
50?
0
300?
150?
0
70?
35?
0
90?
45?
0
Tip234 BaseTip234Base
Tip234 BaseTip234Base
Tip234 Base Tip234 Base
(a)(b)
(c) (d)
(e) (f)