1 Departamento de Inmunología y Oncología, Centro Nacional de
Biotecnología/CSIC, Campus de Cantoblanco, E-28049 Madrid, Spain
2 Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauer
Strasse 108, 01307 Dresden, Germany
Author for correspondence (e-mail:
mtorres{at}cnb.uam.es)
Accepted 11 July 2005
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SUMMARY |
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Key words: Axolotl, Retinoic acid, Vitamin A, Homeobox
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Introduction |
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Urodeles and anuran tadpoles are the only vertebrates able to regenerate
amputated limbs completely. Following Spallanzani's description of this
phenomenon in the 18th century
(Spallanzani, 1768),
scientists have tried to identify the mechanisms underlying limb regeneration
and patterning of the growing blastema. As is the case during limb
development, undifferentiated cells in the regenerating blastema acquire
positional information de novo to reproduce the original pattern. In contrast
to limb development, however, not every positional value needs to be recreated
during regeneration, as the limb amputation can occur at any PD level and the
blastema will reproducibly regenerate only the missing portion. A system of
positional memory recorded in the mature limb is thought to instruct blastema
cells of the exact PD level from which regeneration should take place
(Stocum, 1984
).
Classical experiments showed the role of RA as a major instructor of
positional identity along the regenerating limb PD axis. When anuran tadpole
or urodele limbs are amputated at the wrist and allowed to regenerate in an
excess of RA, a whole PD axis develops rather than just the missing part,
generating a tandem duplication (Maden,
1982; Niazi and Saxena,
1978
). Duplications induced by decreasing amounts of RA result in
increasing distalization of the proximal-most identity of the duplicated
regenerate, showing the ability of RA to change positional values in a
dose-dependent manner (Thoms and Stocum,
1984
).
Cell affinity properties constitute a hallmark of PD positional identity of
blastema cells (Nardi and Stocum,
1983). Consistent with its ability to proximalize regenerating
limbs, RA reprograms blastema cell affinity properties towards a proximal
identity (Crawford and Stocum,
1988
) through the RAR
receptor isoform
(Pecorino et al., 1996
),
downregulates the distal marker Hoxa13
(Gardiner et al., 1995
) and
upregulates the proximal marker Hoxd10
(Simon and Tabin, 1993
).
Recent results shed the first light on the molecular pathways controlling cell
affinity that are activated by RA during limb regeneration. The GPI-anchored
cell surface molecule Prod1 is more abundant in proximal than in distal
blastemas, and is upregulated by RA (da
Silva et al., 2002
). Blocking Prod1 results in loss of the
specific affinity properties of proximal blastemas, whereas overexpression of
Prod1 in limb blastema cells causes them to translocate to more proximal
positions, showing it contributes to the PD affinity code
(Echeverri and Tanaka, 2005
)
Prod1 is also differentially expressed in the mature limb PD axis, suggesting
that it forms part of the positional memory system
(da Silva et al., 2002
).
Some advances in the field of PD specification during limb development in
amniotes came again from analyzing the RA pathway. During embryogenesis, RA is
synthesized throughout the lateral plate mesoderm, but is excluded from the
limb field as limb buds emerge from the trunk
(Berggren et al., 1999;
Niederreither et al., 1997
).
As determined by RA reporter transgene expression
(Reynolds et al., 1991
;
Rossant et al., 1991
), RA
appears to be distributed in a proximal high-distal low gradient in the limb
bud. RA is restricted proximally by limited diffusion from the trunk
RA-producing region and by specific activation of the RA-degrading enzymes
CYP26A1 and CYP26B1 in the distal limb
(MacLean et al., 2001
;
Swindell et al., 1999
). Study
of Raldh2- and CYP26B-deficient mice revealed a role for RA
in promoting limb outgrowth, as well as in limb PD patterning
(Mic et al., 2004
;
Niederreither et al., 2002
;
Yashiro et al., 2004
).
Concurring with the view that RA instructs PD limb cell identities, distal
limb bud region transplants generate proximal structures following RA
treatment, and RA-exposed distal cells contribute to the formation of proximal
skeletal elements (Ide et al.,
1994
; Mercader et al.,
2000
; Tamura et al.,
1997
). As during regeneration, cells of the developing limb bud
display specific affinity according to their position in the PD axis
(Ide et al., 1994
), and RA can
proximalize limb cell affinity in several experimental contexts
(Ide et al., 1994
;
Mercader et al., 2000
;
Tamura et al., 1997
).
Two closely related homeobox genes, Meis1 and Meis2, are
crucial targets activated by RA during PD limb axis patterning
(Mercader et al., 2000;
Yashiro et al., 2004
). Early
subdivision of the limb bud into proximal Meis-positive and distal
Meis-negative domains is necessary for correct PD limb development; ectopic
Meis1 or Meis2 overexpression abolishes distal limb
structures (Capdevila et al.,
1999
), leads to a proximal shift of limb identities along the PD
axis (Mercader et al., 1999
),
and proximalizes distal limb cell affinity properties
(Mercader et al., 2000
).
Meis genes encode transcription factors of the TALE class of homeodomain
proteins. Members of this family share a three-amino-acid loop extension
between homeodomain helices 1 and 2
(Bürglin, 1997). Meis
proteins have an additional conserved N-terminal domain, through which they
interact with members of a second TALE family, the PBC class, encompassing
vertebrate proteins Pbx1 to Pbx4 (Chang et
al., 1997
; Knoepfler et al.,
1997
; Wagner et al.,
2001
). Meis-Pbx dimerization, as well as that of their insect
counterparts, is required for nuclear localization of the proteins
(Abu-Shaar et al., 1999
;
Berthelsen et al., 1999
;
Capdevila et al., 1999
;
Mercader et al., 2000
;
Rieckhof et al., 1997
), and
Meis-Pbx interaction is thus considered to be essential for their
function.
During vertebrate limb regeneration, however, with the exception of Prod1,
the elements in the molecular pathway activated by RA remain unknown. Here, we
use recently developed electroporation methods
(Echeverri and Tanaka, 2003;
Echeverri and Tanaka, 2005
;
Schnapp and Tanaka, 2005
) in
the limb blastema to analyze the role of Meis genes in PD patterning during
regeneration. We provide evidence of a role for Meis genes as crucial targets
of RA proximalizing activity during limb regeneration. Our results show that
the RA-Meis pathway, active during limb development, is essential to specify
RA-induced proximal fates during limb regeneration.
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Materials and methods |
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Cloning axolotl Meis and Pbx genes
Primers used for axolotl Meis1 and Meis2 amplification
from proximal medium bud blastema cDNA (Meis1) and whole tadpole cDNA
(Meis2) were: Meis1, 5'-ACTAGTGATTCTGCACTC, and
5'-CATGTAGTGCCATTGCCCCTC; Meis2, 5'-CTTCAACGAGGACATCGCGGTC and
5'-TTGGGGAATATGCCTCTTTTCTTC. Full-length Meis sequences were
completed by 5' and 3'RACE using the following primers.
For 5'RACE: Meis1, 5'-TGCGATTGGTTAACTAGATGGT and 5'-GATTGGTTAACTAGATGGTGTTAC; Meis2, 5'-AGCGTAGGAACAGGGCGAAGTC and 5'-GCATCACGACAGGCCGCACATC.
For 3'RACE: Meis1, 5'-GTAAGTCAAGGTACACCA and 5'-CCATGGGAGGATTTGTGA; Meis2, 5'-GTGAACCAAGGGGACGGGC and 5'-ACAGCGTGGCCTCTCCTG.
The axolotl Pbx1 full-length sequence was identified in the axolotl EST
sequencing project database (Habermann et
al., 2004). Axolotl Meis1, Meis2 and Pbx1
nucleotide sequences have been submitted to GenBank with accession numbers
DQ100364, DQ100365 and DQ100366.
In situ hybridization
The following probes were used: axoMeis2 (+337 to +828) and
axoHoxa13 (+552 to +834). Whole-mount in situ hybridization was
performed as described (Wilkinson and
Nieto, 1993), with proteinase K treatment (10 µg/ml) for 10
minutes for the youngest embryos and 15 minutes for stage (St) 36 to St43
tadpoles.
For in situ hybridization on sections, 12 µm cryosections were collected
on Superfrost slides, air-dried (2 hours), and hybridized as described
(Myat et al., 1996).
Immunohistochemistry and antibodies
Tissue samples were collected and fixed immediately in 4% paraformaldehyde
(PFA; overnight, 4°C), rinsed in PBS and dehydrated in methanol. Specimens
were rehydrated to PBS, immersed in 30% sucrose in PBS (overnight, 4°C),
OCT-embedded, sectioned (12 µm) on a cryostat and collected on Superfrost
slides. Prior to immunohistochemistry, slides were air-dried (30-60 minutes,
RT), refixed in ice-cold acetone (2 minutes, 4°C), rinsed in PBS, treated
with 100 mM glycine in PBS (30 minutes) and washed in PBT (PBS 0.1% Tween-20).
Sections were permeabilized with 0.05% Triton X-100 in PBS (10 minutes),
followed by a PBT wash (5 minutes). A second permeabilization step was
performed with PBS:DMSO (1:1; 8 min), and a PBT wash. Slides were blocked in
10% goat serum (overnight at 4°C), incubated sequentially with the primary
antibody, with biotin-conjugated secondary antibody and with streptavidin-Cy3
(Jackson) (1 hour each, room temperature).
Anti-Meis-a antibody was generated in rabbits with a synthetic peptide corresponding to the conserved C-terminal domain of Meis1a and Meis2a/Meis2b isoforms (GMNMGMDGQWHYM). Anti-Meis1 and -Meis2 antibodies were raised in rabbits using the peptide sequences HAARSMQPVHHLNHGPP and HAPRPIPPVHHLNHGPP, respectively. Anti-Pbx1/Pbx2/Pbx3/Pbx4 was purchased from Santa Cruz Biotechnology and used at 20 µg/ml. Overexpressed tagged Meis1, Meis2 and Pbx1 proteins were detected using anti-FLAG (Sigma) and anti-Myc monoclonal antibodies.
Quantitative RT-PCR and western blot analysis
For western blot, blastemas were lysed in RIPA and 15 µg protein lysate
was transferred to nylon membranes and counterstained with Ponceau Red to
visualize total protein.
Quantitative PCR was performed on an ABIPrism 7700 thermocycler (Applied Biosystems) applying 1 cycle at 50°C for 2 minutes, 1 cycle at 95°C, 10 minutes, 40 cycles at 95°C, 15 seconds, and at 67°C, 90 seconds, followed by 1 cycle at 95°C, 15 seconds, and at 60°C, 15 seconds, and a final step at 95°C, 15 seconds. For each cDNA sample, two dilutions (1/50 and 1/500) were amplified in duplicate. The following primers were used:
Electroporation of DNA constructs and antisense morpholino into limb blastemas
Axolotl Meis1 and Meis2 cDNA cloned into pCMVTag2b and
axolotl Pbx1 cloned into pCMVTag3b vectors (Stratagene) were diluted
in PBS (0.25 µg/µl), together with a CMV-nuclear EGFP reporter plasmid
(0.05 µg/µl). Using a microinjector (Inject+Matic, Geneva), DNA solution
(7 nl), with Fast Green added to improve monitoring, was injected at
three different blastema sites to ensure homogeneous distribution. Immediately
after injection, five pulses were given (5 mseconds, 150 V/cm each) using flat
circular electrodes (3 mm diameter) with an electroporator (BTX ECM-830, San
Diego). To count GFP-positive cells, limbs were fixed in 4% PFA (overnight,
4°C), soaked in 30% sucrose in PBS (2 hours) and OCT-embedded.
Longitudinal cryosections (30 µm) were collected on slides, air-dried (2-6
hours), fixed in ice-cold acetone (2 minutes), rinsed in PBS and mounted in
anti-fading mounting medium.
Fluorescein-coupled antisense morpholino oligonucleotides (MOs) were designed against 5'-coding regions of axoMeis1 (5'-AAGTCGTCGTACCTTTGCGCCATCG) and axoMeis2 (5'-GGAGTTCATCGTACAGAAACATGAT) (Gene Tools, Corvallis, OR). A fluorescein-coupled standard MO was used as control. MO were diluted in water and stored at 20°C as a 5 mM stock solution. For injection, MO were diluted to 1.5 mM in PBS and heated (68°C) for 10 minutes prior to use. Injection and electroporation were as indicated above for DNA constructs.
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Results |
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An antibody that recognizes an epitope conserved in all short isoforms of
mouse and axolotl Meis proteins (anti-Meis-a) specifically detects a nuclear
signal in the embryonic CNS, in a pattern that correlates with Meis2
mRNA distribution (Fig. 1E-H).
Using this antibody, we examined Meis protein distribution during limb
outgrowth. At stage H36, when the limb bud has just formed, the Meis protein
signal is strong, nuclear and uniformly distributed throughout the limb bud
(Fig. 1I). From stage 40, we
found that the limb bud was subdivided into two regions defined by Meis
protein distribution, a proximal region retaining a strong nuclear Meis-a
signal and a distal region showing a low, diffuse Meis-a signal with
cytoplasmic enhancement (Fig.
1J). During subsequent limb bud stages, we found progressive
growth of the distal, non-nuclear Meis region and maintenance of the proximal
confinement of nuclear Meis expression
(Fig. 1K). In early limb buds,
Meis-a signal was detected in a nuclear pattern in the ectoderm
(Fig. 1I). Ectodermal
expression has been described in the mouse for Meis2
(Oulad-Abdelghani et al.,
1997) and Meis1 (N.M. and M.T., unpublished).
Meis2 mRNA distribution during limb development was uniform with no
proximal restriction at any stage in the mesenchyme, and was weak or absent in
the ectoderm (Fig. 1N-P). These
observations suggest that Meis protein expression could be subject to
post-transcriptional, and/or subcellular distribution, control during limb
development. We observed no expression by in situ hybridization using
different Meis1 probes in the embryo, whereas Meis3 was
detected in the CNS, but was not detected in developing limbs (not shown). We
cannot disregard, however, the fact that technical problems might underlie our
inability to detect Meis1 transcripts in situ, as RT-PCR analysis
indicated the presence of Meis1 mRNA in embryos (data not shown).
Meis1 expression may therefore also contribute to the proximal
enrichment in Meis-a signal, as well as to the ectodermal Meis expression. We
also analysed the expression of Pbx proteins during limb development using an
antibody that recognizes a conserved epitope present in all Pbx proteins
(Pbx1-4). Nuclear Pbx expression was detected ubiquitously along the limb bud
PD axis, with higher expression in the proximal versus distal regions of the
bud (Fig. 1M).
We next tested whether Meis expression was RA-regulated during axolotl limb development. Exposure of stage 43 axolotl larvae to 0.12 mM RA in the swimming water for 22 hours produced an extension of the Meis protein expression domain and an enhancement of its nuclear signal (Fig. 1L), as well as an overall increase in Meis2 mRNA expression (Fig. 1Q).
These results indicate that the proximal restriction of Meis activity during limb development and its positive regulation by the RA pathway are conserved between the axolotl and amniotes.
|
Similar samples were analyzed by western blot to determine protein variations. Meis proteins detected using anti-Meis-a were 4-fold more abundant, and Meis2 was 3-fold more abundant, in proximal versus distal blastemas (Fig. 2B). RA treatment of distal blastemas elevated Meis protein levels over those in proximal blastemas; Meis proteins detected with anti-Meis-a increased 6-fold, whereas Meis2 increased 4.5-fold compared with proximal blastemas (Fig. 2B). In immunohistochemical analysis, an endogenous Meis-a signal was not detected in distal blastemas from untreated animals at any regeneration stage (Fig. 2C,F and not shown); however, a nuclear Meis-a signal was detected in the majority of distal blastema cells three days after RA treatment (Fig. 2D,G). The Meis-a nuclear signal was stronger and more widespread in the blastema 6 days after RA treatment. At this stage, however, a narrow distal Meis-a low-expression area was observed (Fig. 2E,H). The strong Meis-a signal persisted 9 days post-injection in proximal blastema regions (not shown). The Hoxa13 expression pattern was complementary, with broad expression in blastemas from untreated animals, disappearing 3 days after RA treatment and re-appearing at 6 days post-treatment in the Meis-a low-expression area (Fig. 2I-K).
|
Analysis of Meis mRNA or protein levels in tissues from several PD positions of the mature limb showed no differences for Meis1 or Meis2 (not shown). We thus found no direct evidence that Meis activity takes part in positional memory, although we cannot exclude that regulation of Meis activity in the mature limb takes place at the subcellular localization level.
Meis proteins proximalize the affinity properties of limb blastema cells
We used overexpression experiments for functional testing of Meis protein
involvement in PD patterning during limb regeneration. Because Meis function
may require the presence of a Pbx partner, we included co-expression of Meis
together with Pbx1 in our analysis. For these experiments, we used a
full-length Pbx1 cDNA identified in the axolotl cDNA sequencing project
(Habermann et al., 2004).
Expression plasmids driving different Meis or Pbx1 proteins fused to the FLAG
epitope or driving EGFP (Fig.
3A) were electroporated into the limb blastema 6 days after
amputation. When overexpressed without Pbx1, Meis proteins were detected only
until 48 hours after electroporation. By contrast, when introduced together,
both Pbx1 and Meis proteins were detected until 5 days after electroporation
(Fig. 3B). Meis or Pbx1
proteins were no longer detected from day 6, but EGFP persisted up to 20 days
post-electroporation (Fig. 3C
and not shown). The proportion of electroporated cells ranged from 5 to 10% of
blastema cells, precluding the use of this approach to study the consequences
of Meis/Pbx1 overexpression in pattern formation
(Fig. 3B,C). By contrast, the
persistence of EGFP detection allowed us to determine the contribution of
electroporated cell descendants to the regenerated limbs. Proximal blastemas
were electroporated with different combinations of Meis/Pbx1 and EGFP
expression vectors, and their contribution was compared with that of
GFP-only-electroporated control cells. Control cells were widely dispersed
throughout the PD extension of the regenerate
(Fig. 3D-F). By contrast, any
combination containing Meis1a or Meis2a promoted aggregation of electroporated
cells and their preferential location in proximal regenerate regions, both
evident from 3 days after electroporation
(Fig. 3G-I and data not
shown).
To quantify the results, regenerated limbs were sectioned horizontally and positive cells scored in the different PD regions. Any combination containing Meis1a or Meis2a produced a higher frequency of GFP-positive cells in proximal regions compared with controls (Fig. 3J). Whereas positive cell frequency in distal stylopod plus proximal zeugopod was 25% in controls, it was between 53 and 70% in Meis1a- or Meis2a-electroporated blastemas (Fig. 3J). Meis1b isoform activity in this assay was tested by electroporating the murine protein. In contrast to the results for Meis-a, we observed no effect of Meis1b on PD cell distribution (Fig. 3J). Pbx1 alone neither altered the relative PD distribution of electroporated cells nor contributed appreciably to the Meis effect on PD cell distribution (Fig. 3J). This result is consistent with its expression during limb regeneration, where no differential Pbx expression could be detected between proximal and distal blastemas. On the contrary, a homogeneous nuclear Pbx expression pattern in early to medium bud stage blastemas was observed, which did not increase significantly after proximalization of distal blastemas with RA (Fig. 2L-N and data not shown).
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The proximal-preferential distribution of Meis-expressing cells may result from a number of possible cellular effects induced by Meis overexpression, including distal-specific underproliferation, increased death of Meis-electroporated cells, relocation of Meis-expressing cells to proximal limb regions, or combinations of these. Proliferation and cell death rates of electroporated limbs were analyzed at different regeneration stages, with no notable differences between control and experimental conditions, or between proximal and distal blastema regions (data not shown). Undetectable differences in proliferation or cell death rates could nonetheless cause a significant difference in cell number if they accumulated during the 9-day experimental period.
To determine directly whether proximal relocation was a principal cause of the cell redistribution, we set up focal electroporation to target specific regenerate subregions (Fig. 4A,B). Focal electroporation of control plasmid in the distal third of the medium bud limb blastema (closer than 350 µm to the apical ectodermal cap) resulted in GFP-positive cells that contributed almost exclusively to distal regions, with very few regenerates showing GFP-positive cells in zeugopod (2/12) or stylopod (1/12) (Fig. 4I). By contrast, similar electroporation with Meis2a, alone or with Pbx1, resulted in a large proportion of limbs containing GFP-positive cells in zeugopod (20/23) or stylopod (14/23) (Fig. 4E,F,I). Total cell counts in each PD segment in horizontal sections of regenerates showed that Meis2a, alone or with Pbx1, produced proximal relocation of approximately 40% of the electroporated cells (Fig. 4J).
|
The results show that PD cell sorting or migration is the major cause of the proximal-preferential distribution of Meis-overexpressing cells. The ability of Meis to modify limb cell PD affinity during regeneration is active throughout the PD axis of the regenerating limb. Our results suggest a role for Pbx1 in stabilizing Meis proteins and show that Pbx1 expression is not sufficient to promote proximal relocation.
Meis activity is required for RA-induced proximalization of distal blastemas
To study the consequences of Meis knockdown during RA-induced
proximalization of the limb blastema, morpholino oligonucleotides (MO) that
inhibit Meis1 and Meis2 protein expression in vivo
(Fig. 5) were used in
regeneration experiments. Fluorescently labeled MOs electroporated much more
efficiently into blastema cells than did plasmid DNA, with up to 80% of cells
showing a strong signal after electroporation
(Fig. 6A,B). Using this
approach, we determined the consequences of Meis1 and Meis2 knockdown during
limb regeneration. When Meis1 MO (n=20), Meis2 MO (n=20), or
both MOs (n=38), were electroporated into early-bud proximal
blastemas, no alterations in the regeneration pattern were observed (not
shown).
|
Bilateral amputations were performed, and left and right DI scored after regeneration following RA injection. Differences in degree between left and right duplication were determined by calculating the asymmetry index (AI; left DI minus right DI). Positive AI values indicate stronger duplications in the left limb; negative AI values indicate the opposite. When both left and right limbs were electroporated with control MO, deviations from symmetry were minor (never more than one AI unit in a single animal), and were balanced between left and right sides, so that the average AI was 0 (Fig. 6D, Fig. 7A,B). By contrast, when blastemas on one side were electroporated with control MO and those on the opposite with different combinations of Meis MO, less intense duplications were observed in the Meis MO-electroporated limbs in over half of the cases (Fig. 6C,E). This reduction in the DI affected animals showing any degree of duplication on the control side (Fig. 6E and not shown). The average AI was 0.8 for Meis1 and 1.0 for Meis2, when Meis MOs were electroporated on the left side (Fig. 7C,D). When Meis1 and Meis2 MOs were coelectroporated, the average AI variation was 0.8 when introduced on the left and +1 when introduced on the right. In all cases, DI reductions induced by Meis1 and/or Meis2 knockdown in single animals ranged from 1 to 3 duplication units, and affected 26 out of 46 treated animals (Fig. 7).
These results show that Meis knockdown does not result in an altered pattern of normal regeneration but indicate that Meis genes act downstream of RA to induce the proximalization of the regenerating limb.
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Discussion |
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|
During amniote limb development, the limit of Meis gene expression
in the patterned chicken limb corresponds to the boundary between the stylopod
and the zeugopod. Localized inhibition of RA signaling in the chick disrupts
skeletal elements proximal to the elbow/knee, but does not affect regions
distal to this point. Conversely, localized RA excess disrupts elements distal
to the elbow/knee but spares elements proximal to this point
(Mercader et al., 2000). These
results suggested a major subdivision of the limb bud into an RA-Meis domain
that generates regions proximal to the stylopod/zeugopod boundary, and an
RA-Meis-negative area that generates regions beyond this boundary. We found
here that Meis gene expression and regulation through RA is conserved between
amniote and amphibian limb development. Nonetheless, in the chick, RA exposure
or Meis overexpression does not impose a stylopod character, but proximalizes
cells at any distal position in a graded manner
(Mercader et al., 1999
).
Similar results are obtained after RA exposure during limb regeneration;
duplications induced by decreasing amounts of RA result in increasing
distalization of the proximal-most identity of the regenerate
(Thoms and Stocum, 1984
). The
alterations in PD identity promoted by changes in Meis activity seem to follow
similar rules; Meis overexpression proximalizes cell affinity at any position
of the regenerating PD axis, and Meis knockdown distalizes the proximal-most
identity of the regenerate, irrespective of the initial specification status
of the blastema. These observations suggest that positional information is
encoded uniformly, and is recognized continuously along the PD axis during
both limb development and regeneration. In addition, the components of the
positional code appear to remain sensitive to the RA-Meis pathway at any PD
position.
|
Another interesting aspect of limb PD specification mechanisms is the
regulation of cell affinity. PD axial level-specific affinity is a hallmark of
PD identity, and is modulated by RA during development and regeneration. The
fact that Meis rapidly induces cell relocation in the early blastema suggests
that PD-specific affinity is already established at this stage. Cell lineage
tracing and transplantation experiments have also suggested an early PD
sub-division of the limb blastema
(Echeverri and Tanaka, 2005).
Alterations of Meis activity at later stages similarly induced cell
relocation, but within a restricted subregion of the regenerate, suggesting
progressive PD compartmentalization of the blastema, or progressive limitation
in cell migratory ability.
The GPI-anchored Prod1 molecule is downstream of RA in the proximalizing
pathway, at least as part of the PD affinity code
(da Silva et al., 2002;
Echeverri and Tanaka, 2005
).
Prod1 appears to form part of the positional memory system that enables
blastema cells to `know' which limb parts are missing. By contrast, we found
no evidence of Meis genes as part of the positional memory system, suggesting
that the Meis pathway belongs exclusively to the patterning network
re-activated after amputation. In this case, after proximal amputation, the
Meis pathway would be activated by the positional memory system, so that the
initial Prod1 status may determine the level of Meis activation. The answers
to these questions must await isolation of the axolotl Prod1 counterpart.
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ACKNOWLEDGMENTS |
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Footnotes |
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