Developmental Biology 279 (2005) 391 – 401
www.elsevier.com/locate/ydbio
Proximodistal patterning during limb regeneration
Karen Echeverri*, Elly M. Tanaka*
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstra, 108 01307 Dresden, Germany
Received for publication 16 August 2004, revised 20 December 2004, accepted 21 December 2004
Available online 19 January 2005
Abstract
Regeneration is an ability that has been observed extensively throughout metazoan phylogeny. Amongst vertebrates, the urodele
amphibians stand out for their exceptional capacity to regenerate body parts such as the limb. During this process, only the missing portion of
the limb is precisely replaced—amputation in the upper arm results in regeneration of the entire limb, while amputation at the wrist produces
a hand. Limb regeneration occurs through the formation of a local proliferative zone called the blastema. Here, we examine how
proximodistal identity is established in the blastema. Using cell marking and transplantation experiments, we show that distal identities have
already been established in the earliest stages of blastemas examined. Transplantation of cells into new environments is not sufficient to
respecify cell identity. However, overexpression of the CD59, a cell surface molecule previously implicated in proximodistal identity during
limb regeneration, causes distal blastema cells to translocate to a more proximal location and causes defects in the patterning of the distal
elements of the regenerate. We suggest a model for the limb regeneration blastema where by 4 days post-amputation the blastema is already
divided into distinct growth zones; the cells of each zone are already specified to give rise to upper arm, lower arm, and hand.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Regeneration; Limb; Proximodistal patterning; CD59; Urodele
Introduction
During axolotl limb regeneration, amputation along the
proximodistal axis initially results in the formation of a
proliferative zone called the blastema. Subsequently, all the
structures distal to the plane of amputation are regenerated
(Brockes, 1997; Kumar et al., 2000; Stocum and Maden,
1990). How the limb regenerates only the missing part is not
fully understood. It is thought that the cells at the cut surface
retain a positional memory of their origin and this stored
information is used to regenerate only the correct elements.
Transplantation studies have revealed that the blastema
maintains its identity when transplanted into new environments. When, for example, a 10-day limb blastema is
grafted into a fin, it still forms the limb elements it would
* Corresponding authors. Max Planck Institute for Molecular Cell
Biology, Pfotenhauerstra 108, 01307 Dresden, Germany. Fax: +49 351 210
1489.
E-mail addresses: [email protected] (K. Echeverri)8
[email protected] (E.M. Tanaka).
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2004.12.029
have formed originally, implying that all the proximal–distal
patterning information is present within these blastemas
(Stocum, 1984). Several questions arise from such observations. First, would earlier stage blastemas also display this
autonomy upon transplantation? Second, does this autonomus ability to form a blastema represent the capacity to
bself-organizeQ into a limb (Stocum, 1984), or does it mean
that the proximo-distal identity of blastema cells is already
determined by this stage? Related to this, would transplantation of a small group of blastema cells into a new limb
environment cause them to alter their fate?
Several experiments suggest that positional identity in the
limb may be encoded as a proximal to distal gradient of cell
surface molecules. If a distal blastema is grafted onto the
dorsal side of a proximal blastema, then the grafted blastema
is displaced during the course of regeneration to its level of
origin along the proximodistal axis, where it remains and
gives rise to the regenerate it normally would have formed
(Crawford and Stocum, 1988). Other transplantation experiments have shown that if wrist level blastema cells are
grafted onto a shoulder stump, then a normal limb is
392
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
regenerated mainly by growth from the proximal stump and
the wrist cells are pushed down along the axis to form the
hand (Pescitelli and Stocum, 1980). Furthermore, if proximal
and distal blastemas are confronted in a culture dish, the
proximal blastema always engulfs the distal one, an activity
attributed to a gradient of cell adhesivity along the PD axis
(Nardi and Stocum, 1983). These experiments indicate that
proximodistal identity is an intrinsic property of the
blastema, and is manifested on the cell surface.
Retinoic acid and its precursor retinoid are known to be
able to reprogram the positional identity of blastema cells.
When a wrist blastema is exposed to low levels of retinoic
acid, it can regenerate an entire arm (Maden, 1982). The
extent of the proximalization is dose and time dependent.
The blastema is most sensitive to retinoic acid during early
blastema formation when cells are dedifferentiating and
proliferating (Niazi et al., 1985). A major question is how
retinoic acid reprograms positional identity.
Recently, Morais da Silva et al. have provided potential
molecular insight into these questions. They showed that the
GPI-anchored cell surface protein, CD59, was upregulated in
both the mature limb and the limb blastema in response to
retinoic acid. Furthermore, the transcripts are more abundant
in the proximal versus distal limb, suggesting that higher
levels of CD59 are associated with proximal identity. A role
for CD59 in positional identity was indicated by the ability of
anti-CD59 antibodies to block the in vitro engulfing activity
of a proximal blastema on a distal blastema (Morais da Silva
et al., 2002). These in vitro data indicated that the presence of
CD59 is required for this engulfment activity, but the data
did not address whether altering CD59 levels could proximalize blastema cell identity. Furthermore, the relevance of the
engulfment assay to positional identity is not known.
There are still many outstanding questions regarding
limb patterning during regeneration. Does CD59 encode
positional information in the limb? Furthermore, how is
proximodistal patterning achieved in the normal blastema?
In this paper, we show by cell marking and transplantation
that proximodistal specification occurs at an early stage in
blastema formation. We observe the labeling of distinct
zones that will go on to form stylopod, zeugopod, and the
autopod. Furthermore, by developing the ability to overexpress candidate genes in the blastema, we show that
CD59 has the ability to respecify distal blastema cells to
become proximal. Overexpression of CD59 in the early
blastema also has an overall effect on the patterning of the
regenerate resulting in shortening of the radius and ulna and
loss of digits.
Materials and methods
Animals procedures
Axolotls (Ambystoma mexicanum) used in all experiments were bred in captivity in our facility and were
maintained at 17–208C. Animals of between 2 and 4 cm
were used in all experiments. To initiate regeneration,
limbs were amputated at the interface between the radius/
ulna and the metacarpals for wrist blastemas and through
the end of the humerus for upper arm blastemas. For all
experimental procedures, axolotls were anesthetized in
0.01% ethyl-p-aminobenzoate (Sigma).
Electroporation
Limb blastemas were electroporated 3 days postamputation. Animals were anesthetized and immobilized
on an optically clear polymer matrix: sylgard (Dow
Corning). All further procedures were carried out under
an Olympus Stereo SZ microscope. Lineage tracer, 0.5
Ag/Al CMV-EGFP or CMV-DsRed2-N1 was injected
into the limb blastema using a Narishige micromanipulator connected to a World Precision Instruments
pressure injector (model PV820). Five external pulses
were then applied using tweezer electrodes, 50 V, and
50 ms.
Imaging of labeled blastemas
For imaging, the animals were anesthetized, placed on a
coverslip, and imaged using a 10 Plan-Neofluor objective
on a Zeiss Axiovert 2 microscope controlled by a
Metamorph image acquisition system (Visitron) or using
an Olympus Stereo SZX12 microscope with fluorescent
attachment controlled by a Diagnostic Instruments image
acquisition software. Cells were first visible 24 h postelectroporation.
Overexpression of CD59
The plasmid containing CD59 was a kind gift from
Jeremy Brockes and was co-transfected with a nuclear
EGFP plasmid made by Wulf Haubensak (Max Planck,
Dresden) to allow tracing of the transfected cells. The
contralateral limb was used as a control and was transfected
with CMV-DsRed -N1 (Clontech). The limb blastemas were
imaged as described above.
Alcian blue staining of limb regenerates
Limb blastemas were transfected as described above
with CD59 plus CMV-GFP or the contralteral limb was
transfected with CMV-DsRed and CMV-nucGFP. 20
days post-amputation, the regenerates were fixed overnight in 4% PFA and washed in PBS. The limbs were
then dehydrated in an ETOH series, 25, 50, 70, 100%,
10 min each. They were then placed in an alcian blue
solution and placed at 378C until all skeletal elements
were visibly stained, about 24 h. The stained limbs
were then washed in an EtOH/acetic acid mix for 1 h
at room temperature and then gradually rehydrated in an
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
Table 1
BrdU incorporation in electroporated cells
393
Table 2A
Summary of cell marking and transplantation data in the limb blastema
Construct
Number of BrdU
negative cells
Number of
BrdU positive
cells
Number
of cells
counteda
CMV-EGFP
CMV-EGFP + CD59
22 (30%)
85 (92.4%)
55 (70%)
7 (7.6%)
75
92
a
Blastema sections from three different animals were stained with antiBrdU-Rhodamine and imaged. Only cells expressing EGFP plasmid were
counted for BrdU incorporation.
EtOH/PBS series 90, 70, 50, 25%. The limbs were then
put through a glycerol/PBS series 25, 50, 70, 80% and
were finally imaged on an Olympus SZX12 dissecting
microscope.
Transplantation of blastema cells
Cells in a distal or proximal position within the limb
blastema were transfected as described above. Five days
after amputation, the labeled cells were imaged as
described above, the epidermis was peeled back off the
blastema, the area containing the labeled cells was
dissected away from the main blastema using forceps.
The group of labeled cells was then inserted into a
recipient blastema that had been prepared by having the
epidermis peeled back. The donor labeled cells were
placed into the host and the epidermis was replaced, the
wound was allowed to heal and then the animal was
placed into water containing penicillin and streptomycin.
The animals were allowed to recover for 2 days before
being imaged.
Initial position of
marked cells in the
upper arm blastema
Position of marked cells
in the final regenerate
Upper
arm
only
Lower
arm
only
Upper and
lower arm
Hand
Proximal
Distal
7
0
6
0
4
0
No
17
a
Number of
animalsa
17
17
In each category of transplants, all animals showed the phenotype.
PBS plus 5% sucrose and then frozen in TissueTec (OCT
compound, Sakura). Twenty-micrometer longitudinal cryosections were then cut and processed for BrdU staining.
The cryosections were rehydrated in PBST, treated with
2N HCL for 30 min at room temperature and then washed
extensively with PBST (PBS plus 0.1% Tween 20). The
sections were blocked for 1 h in PBST plus 20% goat
serum and 2 mg/ml BSA. Sections were then incubated
overnight in anti-BrdU monoclonal antibody conjugated
with rhodamine (Tanaka et al., 1997), diluted 1:25 in
block solution (see Table 1). The nuclei were stained by
incubating in 1 Ag/ml Hoescht in PBST. The sections
were counted and imaged using a Zeiss Axiovert 2.
The TUNEL assay was carried out on cryosections using
the In Situ Cell Death Detection Kit, TMR red (Roche)
according to manufacturer’s instructions.
Results
BrdU and TUNEL histochemistry
The early blastema contains distinct proximal–distal
domains
For BrdU incorporation experiments, animals with 3or 10-day blastemas were injected intraperitoneally with a
5 mg/ml solution of BrdU and fixed 4 or 12 h later.
Axolotl limbs were fixed overnight in 4% PFA, washed in
We wanted to determine whether distinct proximal–
distal domains were distinguishable early during blastema
formation. If cells were labeled with a fluorescent lineage
tracer in the distal part of an early blastema and a trail of
Fig. 1. Cells in the distal region of an upper arm blastema are fated to form hand structures. (A) Overlay of a fluorescent image with the DIC image of a 4-day
blastema electroporated with CMV-DsRed in the most distal region. (B) By 12 days post-amputation, the distal cells have divided and remained in the most
distal part. (C) At 20 days post-amputation, the whole limb has regenerated and the labeled cells contribute to forming the digits (C). The dashed line marks the
plane of amputation. Scale bar in A, B = 100 Am, C = 500 Am.
394
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
395
Fig. 3. Labeling of middle and distal cells. In the early blastema, the cells lying in the mid zone between the amputation plane and the wound epidermis were
labeled with CMV-nucGFP (A) and the most distal cells were labeled with CMV-DsRed (B), the overlay with the DIC blastema image is seen in panel C. Once
the limb is regenerated, the green cells have proliferated (D) and contributed to forming the lower arm (E), while the distal cells contributed uniquely to forming
the digits (F). Scale bar A, B, C = 100 Am, D–F = 500 Am.
these labeled cells was then observed along the proximal–
distal axis of the regenerate, this result would suggest that
patterning occurs late in regeneration and that early distal
blastema cells have the potential to form both proximal
and distal elements of the regenerate. In contrast, if
labeling cells in the distal blastema would give rise to
only the hand while labeling the proximal blastema cells
would give rise to upper arm elements only, this would
suggest that distinct proximal and distal domains were
present very early during blastema formation.
To distinguish between these possibilities, we electroporated distal versus proximal areas of 3-day blastemas with
expression plasmids encoding GFP or DsRed and then
started imaging live samples 24 h later. For technical
reasons, 3 days post-amputation was the earliest time point
at which only blastema cells could be specifically labeled.
Cells electroporated in limb blastemas retain the GFP or
DsRed proteins for 20–28 days which is longer than we
have seen when spinal cord cells are transfected with the
same plasmids (Echeverri and Tanaka, 2003). This allowed
us to easily track the final position of transfected cells in the
limb regenerates.
When the blastema tip was electroporated at day 3, we
found cells labeled in the early 4-day blastema lying
underneath the wound epidermis (Fig. 1A). When we
imaged the same sample at day 12, just as skeletal
elements begin forming and the blastema has grown
approximately 3 mm in length, it is clear that all the
labeled cells remain in the tip (Fig. 1B). This distal
location was maintained in the 20-day regenerate where
the limb elements were virtually complete (Fig. 1C). In all
cases where cells at the distal tip of the blastema were
labeled, they contributed only to distal elements of the
regenerate (Table 2A).
In order to compare the ultimate fate of distal and
proximal blastema cells, we then labeled two distinct
zones in the same blastema. Cells in the proximal region
of the blastema were labeled with CMV-nucGFP (Figs.
2A, C) and in the distal region with DsRed (Figs. 2B, C).
The two marks were approximately 300 Am apart. After
10 days of regeneration, both groups of cells had
proliferated but had not mixed. Cells from the proximal
region of the 4-day blastema remained in the proximal
region while the distal cells remained at the tip (Figs. 2D–
F). At 18 days of regeneration, limb elements are
differentiating, and the distally labeled cells are associated
with the autopods, and the proximal cells with the forming
stylopod (Figs. 2G–I). The final destiny of the labeled
cells is shown in Fig. 2J, where the proximal cells have
populated the upper arm and the distal cells have
populated the hand. In different samples, cells labeled in
the proximal region of the limb blastema contributed to
Fig. 2. Labeling of proximal and distal cells in an upper arm blastema. (A) A group of cells lying next to the plane of amputation were labeled with CMVnucGFP. (B) The most distal cells of the same blastema were labeled with CMV-DsRed. (C) Overlay of the DIC image with the fluorescent images from A
and B, showing the position of the labeled cells within the blastema. (D, E, F) The same blastema 10 days post-amputation. (G, H, I) 18 days after
amputation, the first morphological signs of differentiation were visible and the marked cells remain segregated in discrete regions of the regenerating limb.
A digit can be seen in I. (J) At 28 days post-amputation, the limb is completely regenerated and the GFP-labeled cells are seen in the upper arm elements
(green cells) while the DsRed labeled cells were found entirely in the digits (red cells). Dashed line marks the plane of amputation. Scale bar A–C = 100 Am,
D–I = 500 Am, J = 100 Am.
396
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
either upper arm only or depending on the size of the
group of cells to both upper and lower arm formation but
in no cases did proximal cells contribute to formation of
the hand (Table 2A). In contrast, when we marked a
middle zone of blastema cells lying approximately 250 Am
proximal to the plane of amputation (Figs. 3B, C), we
observed that the cells contributed to forming only the
lower arm (Figs. 3E, F). These data suggest that proximal
and distal identities may be set up very early in the
blastema and that the 3- to 4-day blastema may be divided
into three discreet regions already specified to form upper
arm, lower arm, and hand.
Fig. 4. Distal wrist blastema cells transplanted into the proximal region of an upper arm blastema contribute to distal limb structures. (A) Distal cells of a wrist
blastema were transfected were CMV-nucGFP. These cells were transplanted into an upper arm blastema to a proximal position lying next to the plane of
amputation (B). (C) By 14 days post-amputation, the cells moved away from the plane of amputation. (D, E) At 22 days after amputation, the cells had
migrated to a distal location and begun to form a digit. (F, G) 28 days post-amputation, the labeled cells had contributed to forming a distally located ectopic
digit (F, G). Scale bar A, B = 100 Am, C, D, F = 500 Am, E, G = 100 Am.
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
The determination of early limb blastema cells
The cell marking experiments indicated that distinct
regions of the early blastema give rise to either proximal
or distal structures. To ascertain if the proximodistal
identity of blastema cells was determined by this stage,
we labeled groups of either distal or proximal cells with
nucGFP or DsRed and then transplanted the groups of
cells to a new location. A group of blastema cells
approximately 400 Am in diameter was transplanted,
usually with between 20 and 50 cells in the clump
surviving the transplantation. When distal blastema cells
from a wrist were transplanted into the most proximal area
of an upper arm blastema (Figs. 4A, B), the cells were
gradually pushed down towards the distal part of the
blastema during the course of regeneration (Fig. 4C).
Eventually, the cells rested in the hand region and began
to differentiate to form a digit (Figs. 4D, E, F, G). These
results imply that distal cells in a very early blastema
already have an identity that cannot be changed based on
transplantation into a new environment. The same result
was seen when distal cells from an upper arm blastema
were transplanted into a proximal location in the upper
arm blastema (Table 2B). The converse experiment of
transplanting proximal cells into a distal blastema was
attempted but in all cases the transplant was immediately
rejected or within 48 h all cells had died. In control
experiments, proximal labeled cells transplanted into the
proximal region of an upper arm blastema contributed
only to the proximal regenerate (Table 2B).
397
limb blastema while the contralateral limb was used as a
control and was transfected with DsRed (Fig. 5). As
expected in the control limb, the distally labeled cells
contributed to the formation of the hand regenerate (Figs.
5A, B, C). Strikingly when CD59 was overexpressed in
distal blastema cells, none of the cells that were initially
located in the distal blastema contributed to the hand.
Rather, all of the cells attained a more proximal position in
the final regenerate (Figs. 5D, E, F). This clear respecification of distal cells by CD59 overexpression is observed in
distal cells from a wrist blastema and from an upper arm
blastema (Table 3).
Cellular consequences of CD59 expression
As part of the phenotypic analysis of CD59, we wanted
to determine whether CD59 expression in blastema affected
cellular properties such as cell proliferation or cell death.
Three- and 10-day blastemas from BrdU-injected animals
were fixed after 4 and 12 h. Under this labeling protocol,
70% of cells transfected with GFP alone incorporated BrdU.
In contrast, 7.6% of GFP + CD59-transfected cells
incorporated BrdU, indicating that cell proliferation was
strongly inhibited by CD59 overexpression (Table 1). The
low level of cells incorporating BrdU when transfected with
CD59 and EGFP may be because approximately 90% of the
cells receive both plasmids during co-transfection. TUNEL
staining of comparable samples showed no increase in
apoptosis in CD59-expressing cells.
Phenotype arising from CD59 overexpression
Overexpression of CD59 respecifies distal cells to a more
proximal location
Morais da Silva et al. implicated CD59 as a candidate
cell surface molecule that mediates proximodistal cell
identity. CD59 is more highly expressed in proximal limb
tissues compared to distal tissues and is upregulated in
response to retinoic acid (Morais da Silva et al., 2002). The
role of CD59 has only been tested in the in vitro blastema
engulfment assay whose relevance to positional identity is
not known. Using our electroporation assay, we examined
whether overexpression of CD59 in blastema cells could reset the positional identity of blastema cells. CD59 was cotransfected with nucGFP in the distal region of the 4-day
Although not clearly visible from the DIC images in
Fig. 5, overexpression of CD59 not only respecified
positional identity of the transfected cells, it also had an
overall effect on the patterning of the regenerated skeletal
elements. Blastemas overexpressing CD59 displayed
defects in the patterning of distal limb elements (Fig. 6).
Alcian blue staining of regenerating limbs 20 days postamputation revealed gross patterning defects such as
shortened or missing radius and ulna (Figs. 6B, D, E,
H), missing digits (Figs. 6B, D, F), and misformed
metacarpals (Figs. 6B, D, E, F). The differing extent of
the patterning abnormalitites observed may be due to the
number of cells initially transfected in the blastema. In
Table 2B
Summary of cell marking and transplantation data in the limb blastema
Type of donor
blastema
Marked cells
position
Type of recipient
blastema
Position of transplanted
cells in recipient
Final position
in regenerate
Number of
animalsa
Wrist
Wrist
Upper arm
Upper arm
Upper arm
Distal
Distal
Proximal
Proximal
Distal
Wrist
Upper arm
Upper arm
Wrist
Upper arm
Distal
Proximal
Proximal
Distal
Proximal
Hand
Hand
Upper arm
Transplant died
Hand
10
8
9
11
7
a
In each category of transplants, all animals showed the phenotype.
398
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
Fig. 5. Overexpression of CD59 in the limb blastema causes translocation to upper arm regions. (A) Early blastema transfected with CMV-DsRed alone in the
most distal part. (B) By 12 days post-amputation, the digits are beginning to form and these cells are partaking in forming the hand. (C) The limb has
completely regenerated and the transfected cells have contributed to forming only the new hand. (D) The distal cells of an early blastema were transfected with
CMV-GFP and CD59. (E) At 12 days post-amputation, the transfected cells are located in the more proximal regions of the forming regenerate. (F) When
regeneration was completed, these labeled cells had contributed to forming upper and lower arm only and not the hand. Dashed line marks the plane of
amputation. Scale bar B, C = 500 Am, A, C, D, F = 100 Am.
contrast, the contralateral control limbs transfected with
CMV-EGFP showed no patterning defects of the skeletal
elements (Figs. 6A, C, E, G).
Discussion
Here, we have investigated proximodistal identity and
patterning during axolotl limb regeneration. We had several
motivations in our experiments. Previous experiments
transplanting whole 10-day blastemas into ectopic locations
showed that blastemas autonomously formed the limb
structures appropriate to their origin. These experiments
indicated that by day 10, the blastema contains all the
information it needs to reform the correct limb elements.
Table 3
CD59 overexpression in the limb blastema
Construct—level
of amputationa
No. of Proximal
Patterning % Which show
animals translocation defectsb
phenotype
CD59—upper arm 12
CD59—wrist
15
CMVGFP alone— 20
upper arm
a
7
8c
0
7
8
0
58
53
0
In all experiments, cells in the distal tip of the blastema were transfected.
Defects in skeletal elements in the regenerated limb analyzed by alcian
blue staining.
c
Translocation into the junction between mature and regenerating tissue
was observed.
b
Day 10, however, is a time when the blastema has likely
undergone considerable patterning events. Furthermore,
such experiments could not indicate what is happening
within the blastema. By labeling small cell groups within
early limb blastemas and by transplantating small cell
groups, we wanted to understand whether the blastema is
subdivided into discrete zones at the earliest stages, and if
cell identity is already determined by this time.
Our cell marking and transplantation experiments
indicate that an upper arm limb blastema is already
divided into distinct proximal–distal zones by 3 days
post-amputation. Furthermore, distal blastema cell identity
is determined by this stage. The 3- to 4-day blastema
represents an extremely early stage of blastema formation
since the axolotl cell cycle is estimated to last between 48
and 72 h so at most 2 cell divisions could have occurred
prior to our cell marking (Maden, 1976). Notably, we
found that labeled cells at the distal tip of 3-day upper arm
blastema, always resulted in a discrete population of hand
cells labeled in the subsequent regenerate. We found no
evidence for a trail of labeled cells along the proximal–
distal axis. These data indicate that the mesenchymal cells
at the distal tip of the blastema is not a growth zone that
will give rise to all limb elements. Furthermore, this
indicates that there is little cell mixing in the normal
blastema after day 3. Indeed, we found that a distally
labeled cell group would often contribute to a single digit
in the regenerate, suggesting that little lateral mixing also
occurs (e.g., see Fig. 3).
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
399
Fig. 6. Overexpression of CD59 causes skeletal patterning defects. Alcian blue staining of regenerated limbs. Panels A, C, E, and G show the control
transfected limbs where the skeletal elements are perfectly formed. Panels B, D, F, and H are examples of the skeletal abnormalities observed due to
overexpression of Cd59, missing digits, shortened radius and ulna, and malformed metacarpals. Scale bar = 500 Am.
We also examined whether the proximodistal identity
of early blastema cells is determined. Transplantation of
small populations of distal cells into proximal regions of a
5-day upper arm blastema translocated distally and gave
rise to hand elements. This behavior is similar to that
found for the transplantation of whole wrist blastemas to
upper arm regions, a phenomenon that was previously
described as daffinophoresisT (Crawford and Stocum,
1988). Whole blastema transplantations always left open
the possibility that a phenomenon such as affinoporesis
was a characteristic of a large population of cells but that
individual cells may behave differently. Although we did
not transplant single cells, our data move toward the
notion that P–D positional identity is maintained as a
stable feature of cell identity on the individual cell basis.
This transplantation data again support the idea that very
early in blastema formation, cell identities are specified
and the action of placing them into a new proximal
environment is not sufficient to reprogram the identity of
the cells.
An intriguing aspect of our results is that proximal
cells transplanted into a distal blastema apparently die.
400
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
This is interesting because cells from the proximal upper
arm must have the capacity to be reprogrammed to give
rise to distal wrist cells during normal regeneration. The
transplantation conditions in these particular experiments
apparently do not support such a transformation. This
opens up the question of whether positional identity of
proximal cells can only become reprogrammed during
the process of dedifferentiation and once cells enter into
the blastema, they become committed to specific
positional coordinates.
CD59 is a cell surface determinant of P–D cell identity
In the distal to proximal transplantations, the baffinityQ of
the transplanted distal cells to the distal region of the host
regenerate indicates the action of a cell surface determinant
in proximodistal patterning. Our data indicate that CD59 is
an important cell surface molecule that mediates this
positional identity and is involved in the overall patterning
of the blastema. Constitutive expression of CD59 in
blastema cells caused a clear proximal shift in their final
position along the limb. Furthermore, we observed a marked
decrease in cell division in CD59 transfected cells. This
inhibition of cell proliferation may play a causal role in the
limb defects observed. An interpretation of the phenotype
would be that constitutive CD59 expression in a significant
population of blastema cells inhibits their ability to divide
and also to contribute to lower arm structures, resulting in
shortening or deletion of the radius and ulna, and reduction
of digits. Taken together, these data suggest that constitutive
CD59 expression causes distal blastema cells to become
locked into a proximal identity and incapable of forming the
lower arm elements and digits. Not all cells in the early
blastema were transfected by the plasmid, presumably
accounting for the formation of some distal elements. So
far, due to technical reasons, we have been unable to
determine if the transfected cells immediately turn on
markers of proximal identity even when they are in a distal
position within the blastema or whether they do so only after
they translocate to a more proximal region.
Implications for limb regeneration models
It is interesting to consider how our data distinguish
between the numerous models proposed for P–D patterning
during regeneration. In such a discussion, it is a given that
our conclusions hold only for blastemas 3–5 days old and
older.
Our cell tracking, BrdU incorporation, and transplantation data appear to eliminate models that postulate end
growth of the blastema. When the tip of an early upper arm
blastema is labeled, the label is exclusively found in the
Fig. 7. Models for proximodistal limb specification during regeneration. Model A: The early blastema is already divided into three zones denoted here in red,
green and yellow. (A) By early blastema stage, 3 days post-amputation, three zones are already set up and the cells contributing to these zones are already
specified to give rise to upper arm, lower arm and the hand. (B, C) As the blastema grows, cells divide within these zones and eventually differentiate into the
lost structures. Model B: Intercalary regeneration model. (D) The early blastema is divided into two zones, proximal and distal, denoted here in red and yellow.
(E, F) The blastema grows by proliferation of the already specified distal cells and by continued contribution of cells from the stump by dedifferentiation; these
cells have a continually more proximal identity and eventually give rise to the lower and upper arm. (G) The blastema cells eventually differentiate and replace
the lost structures.
K. Echeverri, E.M. Tanaka / Developmental Biology 279 (2005) 391–401
hand elements. We have never observed a trail of cells
spanning proximal and distal elements. Our BrdU labeling
experiments also confirm previous observations that cell
proliferation is uniform throughout the blastema (Maden,
1976). Transplantation of distal cells into proximal regions
was not able to respecify their fate. Thus, models where
proximal elements are formed before distal elements from
the same source population seem unlikely. Our results also
eliminate the possibility that a morphogen controls P–D
identity in the 4-day blastema, where distal cells can be
respecified to a proximal fate by exposure to higher
concentrations of morphogen (Wolpert, 1969). However,
as morphogen gradients act over short ranges, our data do
not rule out the possibility that a gradient exists in the earlier
blastema that specifies proximodistal identity.
Our results strongly point to two scenarios for early
blastema patterning. In one model, the early, 4-day blastema
is already divided into three distinct zones that are specified
to become upper arm, lower arm, or hand (Fig. 7, model A).
These cells proliferate and eventually differentiate to form
upper arm, lower arm, or hand. Such a model accounts for
the tendency in our marking experiments to label discrete
limb elements (Table 2A). This model is similar to one put
forth by Dudley et al. to describe how the chick limb bud
may be patterned (Dudley et al., 2002).
Our results could also be consistent with an intercalation
type of model where after amputation in the upper arm, the
first blastema cells are distalized to become hand cells.
Subsequently, the upper arm cells dedifferentiate to generate
blastema cells with intermediate lower limb identities (Fig.
7, model B). This phenomenon is often called intercalation
(Maden, 1980; Stocum, 1984). Such a model could fit with
our data where tip blastema cells always give rise to hand
elements, while cells marked in the proximal regions of the
blastema can give rise either to upper arm only or to upper
arm and lower arm. Many models emphasize the importance
of local cell–cell interactions to promote intercalation.
Although our experiments do not address this issue in
detail, the observation that transplanted distal cells have
affinity for distal limb elements, and the strong role of the
cell surface CD59 in determining positional identity lend
support to such a notion. We could not determine whether
the transplanted distal cells caused neighboring proximal
cells from the host tissue to change their fate into
intermediate positional values, thus limiting our ability to
test the intercalation model.
A possible means of specifying positional information
would be through a gradient of CD59 expression within the
blastema. This could be achieved by downregulating CD59
in dedifferentiating limb cells that would then acquire a
distal identity as they contribute to the blastema. Therefore,
401
a gradient would be produced with the cells in the stump
maintaining the highest level of CD59 and the cells in the
blastema having a lower level of CD59. It will be interesting
to determine whether and how CD59 is downregulated in an
upper arm blastema to generate cells with lower arm and
hand identities.
Acknowledgments
We thank Heino Andreas for dedicated axolotl care and
Jeremy Brockes and Andrew Oates for helpful reading of
the manuscript.
References
Brockes, J.P., 1997. Amphibian limb regeneration: rebuilding a complex
structure. Science 276, 81 – 87.
Crawford, K., Stocum, D.L., 1988. Retinoic acid proximalizes levelspecific properties responsible for intercalary regeneration in axolotl
limbs. Development 104, 703 – 712.
Dudley, A., Ros, M., Tabin, C., 2002. A re-examination of proximodistal
patterning during vertebrate limb development. Nature 418, 539 – 544.
Echeverri, K., Tanaka, E.M., 2003. Electroporation as a tool to study in
vivo spinal cord regeneration. Dev. Dyn. 226, 418 – 425.
Kumar, A., Velloso, C.P., Imokawa, Y., Brockes, J.P., 2000. Plasticity of
retrovirus-labelled myotubes in the newt limb regeneration blastema.
Dev. Biol. 218, 125 – 136.
Maden, M., 1976. Blastemal kinetics and pattern formation during
amphibian limb regeneration. J. Embryol. Exp. Morphol. 36, 561 – 574.
Maden, M., 1980. Intercalary regeneration in the amphibian limb and the
rule of distal transformation. J. Embryol. Exp. Morphol. 56, 201 – 209.
Maden, M., 1982. Vitamin A and pattern formation in the regenerating
limb. Nature 295, 672 – 675.
Morais da Silva, S., Gates, P., Brockes, J.P., 2002. The Newt ortholog of
CD59 is implicated in proximodistal identity during amphibian limb
regeneration. Dev. Cell 3, 547 – 555.
Nardi, J.B., Stocum, D.L., 1983. Surface properties of regenerating limb
cells: evidence for gradation along the proximodistal axis. Differentiation 25, 27 – 31.
Niazi, I., Pescitelli, M., Stocum, D., 1985. Stage-dependent effects of
retinoic acid on regenerating urodele limbs. W. Roux’s Arch. Dev. Biol.
194, 355 – 363.
Pescitelli Jr., M.J., Stocum, D.L., 1980. The origin of skeletal structures
during intercalary regeneration of larval Ambystoma limbs. Dev. Biol.
79, 255 – 275.
Stocum, D.L., 1984. The urodele limb regeneration blastema. Determination and organization of the morphogenetic field. Differentiation 27,
13 – 28.
Stocum, D.L., Maden, M., 1990. Regenerating limbs. Methods Enzymol.
190, 189 – 201.
Tanaka, E.M., Gann, A.A., Gates, P.B., Brockes, J.P., 1997. Newt myotubes
reenter the cell cycle by phosphorylation of the retinoblastoma protein.
J. Cell Biol. 136, 155 – 165.
Wolpert, L., 1969. Positional information and the spatial pattern of cellular
differentiation. J. Theoret. Biol. 25, 1 – 47.