DEVELOPMENT AND STEM CELLS
RESEARCH REPORT
513
Development 140, 513-518 (2013) doi:10.1242/dev.081752
© 2013. Published by The Company of Biologists Ltd
Connective tissue cells, but not muscle cells, are involved in
establishing the proximo-distal outcome of limb
regeneration in the axolotl
Eugen Nacu1,2,*, Mareen Glausch1, Huy Quang Le1, Febriyani Fiain Rochel Damanik1, Maritta Schuez1,
Dunja Knapp1,2, Shahryar Khattak1,2, Tobias Richter2 and Elly M. Tanaka1,2,*
SUMMARY
During salamander limb regeneration, only the structures distal to the amputation plane are regenerated, a property known as the
rule of distal transformation. Multiple cell types are involved in limb regeneration; therefore, determining which cell types
participate in distal transformation is important for understanding how the proximo-distal outcome of regeneration is achieved.
We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, which
can act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule of
distal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connective
tissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of regeneration and are likely to guide
muscle patterning. Moreover, we show that, similarly to limb development, muscle patterning in regeneration is influenced by bcatenin signalling.
INTRODUCTION
Limb development and regeneration involve the coordinated growth
and patterning of several tissues, including bone, soft connective
tissue, muscle, epidermis, peripheral nerve and blood vessels. During
limb development, lateral plate mesoderm-derived cells can form
recognisable limb segments in the absence of muscle formation and
it has been proposed that they are a dominant cell type directing limb
patterning (Christ et al., 1977; Grim and Wachtler, 1991; Kardon et
al., 2002; Kardon et al., 2003; Hasson et al., 2010; Mathew et al.,
2011). Molecular factors that influence the formation of the three
distinct limb segments (upper arm, lower arm and hand), such as
MEIS and HOX genes, are expressed in multiple limb progenitor cell
types, including cells derived from lateral plate mesoderm, as well
as myogenic cells derived from somitic mesoderm (Yamamoto et al.,
1998; Hashimoto et al., 1999; Mercader et al., 1999; Cooper et al.,
2011; Roselló-Díez et al., 2011). These factors probably exert their
patterning influence via expression in the lateral plate mesoderm
derivatives. Their expression in myogenic cells has been linked to
myogenic differentiation, although an influence on muscle patterning
has not been excluded (Knoepfler et al., 1999; Yamamoto and
Kuroiwa, 2003; Heidt et al., 2007).
Salamander limb regeneration provides another context in which
to study specification of proximo-distal limb identity. Upon limb
amputation anywhere along the limb axis, only the missing portion
of the limb distal to the amputation plane regenerates. Furthermore,
when a hand blastema was transplanted to an upper arm stump, a
normal limb regenerated. Examination of melanocytes or labelled
1
Center for Regenerative Therapies Dresden, Fetscherstraße 105, 01307, Dresden,
Germany. 2Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstraße 108, 01307, Dresden, Germany.
*Authors for correspondence ([email protected]; [email protected])
Accepted 23 November 2012
cartilage nuclei indicated that the hand blastema cells contributed
only to the hand, and the lower arm was formed from the upper
arm stump cells through a phenomenon called intercalation
(Stocum, 1975; Maden, 1980; Pescitelli and Stocum, 1980). This
and other transplantation experiments showed that, as a whole, a
limb blastema is autonomously specified to form the limb
structures distal to its site of origin (for review, see Nacu and
Tanaka, 2011). This is termed the rule of distal transformation.
The limb blastema is also composed of lineage-restricted
progenitors (Kragl et al., 2009). Therefore, it is important to know
which cells obey the rule of distal transformation and thus
determine the proximo-distal outcome of regeneration, a crucial
aspect of regenerating a properly patterned limb. Kragl and
colleagues (Kragl et al., 2009) showed that upper limb blastema
cells show heterogeneity in the nuclear expression of transcription
factors associated with limb patterning. Cartilage-derived and
muscle-derived blastema cells expressed MEIS, HOXA9 and
HOXA13, as found for limb development, but Schwann cells did
not. Interestingly, the cellular behaviour of cartilage-derived cells
versus Schwann cells correlated with expression of these factors:
cartilage-derived cells obeyed the rule of distal transformation,
whereas Schwann cells did not. In addition to understanding which
cells obey the rule of distal transformation, it is also important to
determine the molecular mechanisms implemented in patterning
during limb regeneration, and to what extent they are common to
those used during development.
Here, we investigate the role of connective tissue (CT) cells,
which derive from lateral plate mesoderm, and muscle cells in
patterning during limb regeneration by asking whether they obey
the rule of distal transformation. We find that CT-derived blastema
cells display nuclear MEIS in upper arm, but not lower arm or hand
blastemas. At a cellular level, CT-derived blastema cells obey the
rule of distal transformation in the intercalation assay. By contrast,
nuclear MEIS signal is not restricted to upper arm blastemas in
myogenic blastema cells. At a cellular level, myogenic cells break
DEVELOPMENT
KEY WORDS: Limb, Regeneration, Proximo-distal, Muscle, Connective tissue, MEIS
RESEARCH REPORT
the rule of distal transformation. These data indicate that, similarly
to limb development, lateral plate mesoderm-derived cells probably
play a dominant role in patterning of distal structures, whereas
myogenic cells follow the patterning cues of lateral plate
mesoderm-derived cells. We highlight an additional parallel to limb
development by showing that b-catenin activity influences muscle
patterning.
MATERIALS AND METHODS
Labelling of cell types
Ambystoma mexicanum (axolotl) expressing GFP in connective tissue cells
(GFP-CT) or muscle cells (GFP-M) were generated by embryonic
transplantation as previously described (Kragl et al., 2009; Nacu et al.,
2009) (supplementary material Fig. S1).
Transgenics
SceI-CarAct:GFP plasmid (kind gift of Hajime Ogino, Nara Institute of
Science and Technology, Japan) was injected into axolotl eggs as
previously described (Khattak et al., 2009).
Adult surgery
All animal procedures were carried out in accordance with the laws and
regulations of the State of Saxony. Animals were anesthetised in 0.03%
benzocaine (Sigma) prior to surgery. Intercalation assays were performed
as previously described (Maden, 1980). Blastemas, 7 or 10 days post
amputation (dpa), were transplanted on animals 2.5-3.0 cm and 3.0-4.0 cm
in length (snout to cloaca), respectively. Blastemas were transplanted onto
hosts amputated at mid-upper arm or shoulder. Animals were covered by
benzocaine-soaked tissues for 1 hour and afterwards transferred into tap
water. Only animals that regenerated distinct upper arm, lower arm and
hand were analysed.
Sample collection and immunohistochemistry
Samples were fixed in MEMFA, embedded in Tissue-tek (O.C.T. compound,
Sakura) or 7.5% gelatine (Sigma), sectioned at 10 µm thickness and stained
with antibodies (supplementary material Table S1) as previously described
(Mchedlishvili et al., 2007; Mchedlishvili et al., 2012).
For MEIS/PAX7 double staining, the slides were first incubated with
anti-MEIS, then with Fab goat anti-mouse, and finally with Alexa647conjugated Fab rabbit anti-goat. Slides were then blocked with 20% goat
serum in PBS and then incubated with anti-PAX7, followed by Cy3conjugated donkey anti-rabbit.
b-Catenin electroporation
Upper arm blastemas (6 dpa) were injected with 2.5 mg/ml pCS2+ plasmid
expressing a constitutively active form of b-catenin (kind gift of Gilbert
Weidinger, Universität Ulm, Germany) or 2.5 mg/ml CAGGS:GFP as
control. CAGGS:mCherry was added to each mix (final concentration 0.25
mg/ml). Limbs were electroporated at 300 V/cm, 50 ms pulse length, 5
pulses on a BTX-ECM-830 electroporator.
Whole-mount staining of electroporated limbs
Fixed limbs were stained with mouse anti-MHC antibody, then Cy3coupled Fab goat anti-mouse and cleared as previously described (Ertürk
et al., 2012).
Imaging and image processing
Whole-limb images were acquired on Olympus SZX-16. Whole-mount
stainings and limb sections were imaged on Zeiss AxioImagerZ.2
LSM780, Zeiss AxioObserverZ.1 or Olympus-BX61VS. Images were
processed with Fiji software for better visualisation and Volocity software
(PerkinElmer) for 3D reconstruction.
RESULTS AND DISCUSSION
Connective tissue cells obey the rule of distal
transformation
In this study, we were interested in elucidating whether myogenic
and connective tissue (CT)-derived blastema cells, both expressing
Development 140 (3)
MEIS and HOXA13, obey the rule of distal transformation, which
is linked to the proximo-distal outcome of regeneration. In
development, lateral plate mesoderm-derived cells are sufficient for
forming a properly patterned limb (Christ et al., 1977; Grim and
Wachtler, 1991). Therefore, we postulated that during regeneration
CT cells, which are derived from lateral plate mesoderm, obey the
rule of distal transformation.
To test this hypothesis, we first investigated whether MEIS,
which can act as an upper arm identity regulator (Capdevila et al.,
1999; Mercader et al., 1999; Mercader et al., 2005), is present in
nuclei of CT cells only upon upper arm amputation. We generated,
by embryonic transplantation, animals in which the vast majority
of CT cells constitutively express GFP (GFP-CT) (supplementary
material Fig. S1). In 10 dpa upper arm blastemas of GFP-CT limbs,
we identified that 37-62% of GFP+ cells have nuclear MEIS, in
contrast to 0.6% of GFP+ cells having nuclear MEIS+ in 10 dpa
lower arm blastemas (Fig. 1A-F,M-Q; supplementary material
Table S2). These results suggested that connective tissue cells
could indeed obey the rule of distal transformation during
regeneration.
To address the question at a cellular level, we set up the
intercalation assay as previously described: a wrist blastema was
transplanted onto an upper arm stump (Fig. 2A,C,E). If cells from
the grafted blastema end up more proximally of their origin, those
cells break the rule of distal transformation. When wrist blastemas
from GFP-CT animals were transplanted onto upper arm stumps,
the vast majority of GFP+ cells homed to the hand (Fig. 2C,D;
supplementary material Fig. S2). In control experiments of upper
arm GFP-CT blastema transplantations, GFP+ cells were found in
upper arm, lower arm and hand (Fig. 2E,F; Table 1). These results
show that CT-derived blastema cells obey the rule of distal
transformation.
Myogenic cells break the rule of distal
transformation
Next, we investigated whether myogenic blastema cells display
nuclear MEIS signal only upon upper arm amputation by using
animals in which myogenic somitic mesoderm derivatives
constitutively express GFP (GFP-M) (supplementary material Fig.
S1). We found that, in contrast to CT cells, myogenic blastema
cells have nuclear MEIS after amputation through any limb
segment: mid-upper arm (Fig. 1R-V), mid-lower arm (Fig. 1G-L),
and metacarpals (supplementary material Fig. S3). Specifically, 5180% of GFP+ myogenic blastema cells have nuclear MEIS in 10
dpa upper arm blastemas, and 23-86% in 10 dpa lower arm
blastemas (supplementary material Table S2). Because myogenic
blastema cells express PAX7 (supplementary material Table S2)
(Kragl et al., 2009), we explored whether PAX7+ myogenic
progenitors have nuclear MEIS. Double staining of MEIS and
PAX7 revealed that 36.8-91.4% of PAX7+ cells in the blastema are
MEIS+ (supplementary material Fig. S4, Table S3). These results
suggested that during regeneration, myogenic blastema cells have
a different proximo-distal determination system from connective
tissues.
To follow muscle cells in the intercalation assay, we transplanted
wrist blastemas from germline CarAct:GFP transgenic animals,
which express GFP in muscle fibres, onto upper arm stumps
(Mohun et al., 1986). Upon regeneration, GFP+ muscle fibres were
observed in the upper arm, lower arm and hand, indicating that
myogenic blastema cells break the rule of distal transformation
(Fig. 2A,B; Table 1). GFP+ muscle fibres were observed in all
muscles of the upper arm. The humeroantebrachialis and
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Cells in distal transformation
RESEARCH REPORT
515
anconaeus humeralis medialis, which originate in the humerus
(Walthall and Ashley-Ross, 2006), had the highest abundance of
GFP+ muscle fibres (supplementary material Fig. S5, Tables S4,
S5).
Muscle fibres form by fusion of precursor cells; thus, the
observed proximalisation could be the result of fusion of labelled
myogenic blastema cells in the grafted blastema with pre-existing
muscle fibres in the stump. To account for this, we examined
whether satellite cells (mononucleate myogenic cells) were also
proximalised. Upper arms of regenerates that were generated by
transplanting CAGGS:GFP wrist blastemas (in which all cells
express GFP) onto upper arm stumps, were analysed with an
antibody against PAX7, which marks satellite cells. PAX7+GFP+
cells were present in the upper arms of the regenerates (seven out
of seven limbs), indicating that myogenic blastema cells gave rise
to proximalised satellite cells in the intercalation assay (Table 1;
supplementary material Fig. S5, Table S5). These results show that
myogenic cells break the rule of distal transformation.
Our results indicate that connective tissue cells, but not myogenic
cells, guide the proximo-distal outcome of regeneration. In this
regard, patterning in regeneration might recapitulate development,
during which limb bud cells of lateral plate mesoderm origin direct
DEVELOPMENT
Fig. 1. Connective tissue (CT)-derived
blastema cells have nuclear MEIS only
upon upper arm amputation, whereas
myogenic blastema cells have nuclear
MEIS upon upper and lower arm
amputation in axolotl. (A-F) In 10 dpa
lower arm blastemas, CT-derived cells
(green) have no nuclear MEIS (red). (B)
Enlargement of the boxed area in A
showing that MEIS+ cells are surrounded
by GFP+ cells, but do not colocalise. (C-F)
Enlargements of the boxed area in B;
yellow arrowheads indicate MEIS+GFP–
cells. (G-L) In 10 dpa lower arm blastemas,
myogenic cells (green) have nuclear MEIS
(red). H is an enlargement of the boxed
area in G. (I-L) Enlargements of the boxed
area in H; white arrowheads indicate
MEIS+GFP+ cells. (M-V) In 10 dpa upper
arm blastemas, CT-derived cells (M-Q,
green) and myogenic cells (R-V, green)
show nuclear MEIS (red). (N-Q)
Enlargements of the boxed area in M. (S-V)
Enlargements of the boxed area in R.
White arrowheads indicate MEIS+GFP+
cells. (W) Percentage of myogenic or CT
cells that are MEIS+ in 10 dpa lower arm
blastemas. Number of limbs counted:
dermis, n=5; muscle, n=5 (supplementary
material Table S2). (X) Percentage of
myogenic or CT-derived cells that are
MEIS+ in 10 dpa upper arm blastemas.
Number of limbs counted: dermis, n=3;
muscle, n=2. Scale bars: in A,G,M,R, 1
mm; in B,H, 200 m; in C-F,I-L, 50 m, in
N-Q,S-V, 100 m.
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Development 140 (3)
RESEARCH REPORT
Fig. 2. Connective tissue cells, but not myogenic
cells, obey the rule of distal transformation in
axolotl limb regeneration. (A) Schema of wrist
blastema transplantation from an animal that
expresses GFP in muscle fibres (green) onto a nontransgenic upper arm (UA) stump. (B) Experimental
result of A: upon regeneration, GFP-muscle fibres
(green) are found in the UA, lower arm (LA) and hand.
(C,D) Upon wrist blastema transplantation from a GFPCT animal, in which connective tissue cells express
GFP, onto a non-transgenic UA stump, GFP+
connective tissue cells are found only in the hand of
the regenerate. (E,F) UA blastema transplantation
from a GFP-CT animal onto a non-transgenic UA
stump results in GFP-connective tissues cells in UA, LA
and hand of the regenerate. Dashed lines in B,D,F
outline the limb. Scale bars: 2 mm.
the migration and patterning of naive myogenic progenitors (see
Introduction). Next, we were interested in uncovering the molecules
that might play a role in patterning muscle cells.
b-Catenin signalling is involved in muscle
patterning during regeneration
In limb development, TCF4 of the WNT/b-catenin pathway is
expressed in early muscle connective fibroblasts and plays a crucial
role in dictating the placement of limb muscle masses (Kardon et
al., 2003; Mathew et al., 2011). Kardon and colleagues (Kardon et
al., 2003) showed that expression of constitutively active b-catenin
in chicken limb buds gives rise to ectopic muscle formation. We
were therefore interested to determine whether b-catenin signalling
affects muscle patterning in axolotl limb regeneration.
We overexpressed constitutively active b-catenin (Yost et al.,
1996) in axolotl blastemas using electroporation. Five out of 11
limbs electroporated with b-catenin showed ectopic muscle
formation in the regenerated upper arm, such as on the dorsal side
of the limb close to the triceps (Fig. 3; supplementary material Fig.
S6). Control GFP-electroporated regenerates showed defects in
musculature, as occurs normally during regeneration (Diogo et al.,
2013), but we did not observe in any of the ten samples ectopic
muscle formation similar to that seen in b-catenin-electroporated
limbs.
Table 1. Muscle fibres and satellite cells, but not connective tissue cells, are found more proximal of their origin when a wrist
blastema is transplanted onto an upper arm stump
Cell type followed
Donor blastema origin*
Total transplantations (n)
Upper arm
Lower arm
Hand
GFP+ muscle fibres‡
Wrist
Upper arm
Wrist
Upper arm
Wrist
Upper arm
12
10
7
5
8
6
11
10
7
5
1
3
12
10
n.a.
n.a.
1
6
12
10
n.a.
n.a.
8
6
GFP+ satellite cells§
GFP+ connective tissue cells¶
*The donor blastemas were grafted onto non-transgenic upper arm stumps.
‡
Muscle fibres were followed by transplanting CarAct:GFP blastemas.
§
Satellite cells were followed by transplanting CAGGS:GFP blastemas.
¶
Connective tissue cells were followed by transplanting GFP-CT blastemas.
n.a., origin of satellite cells in the lower arm or hand was not analysed.
DEVELOPMENT
Number of regenerates with GFP+ cells in the limb segments
Cells in distal transformation
RESEARCH REPORT
517
Fig. 3. Overexpression of constitutively active bcatenin induces ectopic muscle formation in
axolotl limb generation. (A,B) Maximum intensity
projection (MIP) from z-stacks through ventral (A) and
dorsal (B) halves of a control electroporated limb. (C,D)
MIP from z-stacks through ventral (C) and dorsal (D)
halves of two example limbs electroporated with an
expression plasmid for constitutively active b-catenin.
Red arrowheads point to ectopic muscles. Muscles
normally present in the upper arm are: triceps branchii
group, green; coracobrachialis longus (CBL), blue;
humeroantebrachialis (HAB), yellow; supracoracoideus
(SC), purple. Scale bars: 1 mm.
Acknowledgements
We thank Heino Andreas, Sabine Mögel and Beate Gruhl for axolotl care. We
acknowledge Hajime Ogino for CarAct:GFP transgenesis construct and Gilbert
Weidinger for the b-catenin construct. We thank Walter Bonacci and Shirley
Galbiati for advice on the manuscript. We apologise for the limitation in
possibility to extensively discuss and cite the literature on patterning during
limb development and regeneration.
Funding
This research was funded by grants from the German Research Foundation
(DFG) [DFG TA 274/3-1, DFG TA 274/3-2, DFG TA 274/3-2]; the Volkswagen
Foundation [I/85018]; and funds from Max Planck Society and the Center for
Regenerative Therapies Dresden [to E.M.T.].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.081752/-/DC1
References
Alvares, L. E., Schubert, F. R., Thorpe, C., Mootoosamy, R. C., Cheng, L.,
Parkyn, G., Lumsden, A. and Dietrich, S. (2003). Intrinsic, Hox-dependent
cues determine the fate of skeletal muscle precursors. Dev. Cell 5, 379-390.
Brand-Saberi, B., Gamel, A. J., Krenn, V., Müller, T. S., Wilting, J. and Christ,
B. (1996). N-cadherin is involved in myoblast migration and muscle
differentiation in the avian limb bud. Dev. Biol. 178, 160-173.
Capdevila, J., Tsukui, T., Rodríquez Esteban, C., Zappavigna, V. and Izpisúa
Belmonte, J. C. (1999). Control of vertebrate limb outgrowth by the
proximal factor Meis2 and distal antagonism of BMPs by Gremlin. Mol. Cell 4,
839-849.
Christ, B., Jacob, H. J. and Jacob, M. (1977). Experimental analysis of the origin
of the wing musculature in avian embryos. Anat. Embryol. 150, 171-186.
Cooper, K. L., Hu, J. K.-H., ten Berge, D., Fernandez-Teran, M., Ros, M. A.
and Tabin, C. J. (2011). Initiation of proximal-distal patterning in the vertebrate
limb by signals and growth. Science 332, 1083-1086.
Diogo, R., Nacu, E. and Tanaka, E. M. (2013). Is salamander limb regeneration
perfect? Anatomical and morphogenetic analysis of forelimb muscle
regeneration in GFP-transgenic axolotls as a basis for regenerative,
developmental and evolutionary studies. J. Anat. (in press).
Ertürk, A., Mauch, C. P., Hellal, F., Förstner, F., Keck, T., Becker, K., Jährling,
N., Steffens, H., Richter, M., Hübener, M. et al. (2012). Three-dimensional
imaging of the unsectioned adult spinal cord to assess axon regeneration and
glial responses after injury. Nat. Med. 18, 166-171.
Greig, S. and Akam, M. (1993). Homeotic genes autonomously specify one
aspect of pattern in the Drosophila mesoderm. Nature 362, 630-632.
Grim, M. and Wachtler, F. (1991). Muscle morphogenesis in the absence of
myogenic cells. Anat. Embryol. 183, 67-70.
Hashimoto, K., Yokouchi, Y., Yamamoto, M. and Kuroiwa, A. (1999). Distinct
signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle
precursor and mesenchyme of the chick limb bud. Development 126, 27712783.
Hasson, P., DeLaurier, A., Bennett, M., Grigorieva, E., Naiche, L. A.,
Papaioannou, V. E., Mohun, T. J. and Logan, M. P. O. (2010). Tbx4 and tbx5
acting in connective tissue are required for limb muscle and tendon patterning.
Dev. Cell 18, 148-156.
DEVELOPMENT
Conclusions
We show through intercalation assays that myogenic cells break the
rule of distal transformation, whereas connective tissue cells obey
it. These results suggest that, similarly to development, the
connective tissue cells guide muscle patterning during regeneration.
Therefore, it will be important in the future to study connective
tissue cells in order to understand how the proximo-distal outcome
of regeneration is established.
We show that myogenic blastema cells break the rule of distal
transformation and display nuclear MEIS, which can functionally
proximalise blastema cells (Mercader et al., 2005). We also found
myogenic cells expressing HOXA11 and HOXA13 in 10 dpa lower
arm blastemas (data not shown). It is possible that MEIS, HOXA11
and HOXA13 play a role in patterning of myogenic cells during
regeneration; however, their expression is regulated by signals that
myogenic cells receive from connective tissue cells. For example,
in chicken, HOXA10 predisposes, in an autonomous way, the
myogenic progenitors in the somite to a limb fate (Alvares et al.,
2003). In Drosophila, the identity of muscle cells in different larval
segments is believed to be the result of integration of extrinsic cues
with autonomous expression of HOX homologues (Greig and
Akam, 1993; Roy and Vijayraghavan, 1997).
Alternatively, MEIS and HOX genes might play a role not
related to positional identity in myogenic cells. MEIS, in complex
with PBX, binds DNA cooperatively with MYOD and marks genes
for activation by MYOD (Knoepfler et al., 1999; Heidt et al.,
2007). Also, HOXA11 and HOXA13 have been shown to repress
MYOD and maintain the myogenic progenitors in a proliferative
state during limb development (Yamamoto and Kuroiwa, 2003).
Therefore, it is possible that MEIS, HOXA11 and HOXA13 are not
involved in patterning, but have a role only in proliferation and
differentiation of myogenic cells during regeneration. To
distinguish between these hypotheses, it will be important to
knockdown MEIS and assess the effect on muscle cell
proximalisation in regeneration.
We further show that, similarly to development, activation of
the b-catenin pathway influences muscle patterning, indicating
that molecules involved in muscle patterning are likely to be
conserved between regeneration and development. In addition to
TCF4/b-catenin, TBX4 and TBX5 non-autonomously influence
limb muscle patterning during mouse limb development (Hasson
et al., 2010). Brand-Saberi and colleagues (Brand-Saberi et al.,
1996) previously suggested that N-cadherin might participate in
myoblast path finding. It will be interesting to investigate
whether these molecules play a role in muscle patterning during
regeneration also.
RESEARCH REPORT
Heidt, A. B., Rojas, A., Harris, I. S. and Black, B. L. (2007). Determinants of
myogenic specificity within MyoD are required for noncanonical E box binding.
Mol. Cell. Biol. 27, 5910-5920.
Kardon, G., Campbell, J. K. and Tabin, C. J. (2002). Local extrinsic signals
determine muscle and endothelial cell fate and patterning in the vertebrate limb.
Dev. Cell 3, 533-545.
Kardon, G., Harfe, B. D. and Tabin, C. J. (2003). A Tcf4-positive mesodermal
population provides a prepattern for vertebrate limb muscle patterning. Dev. Cell
5, 937-944.
Khattak, S., Richter, T. and Tanaka, E. M. (2009). Generation of transgenic
axolotls (Ambystoma mexicanum). Cold Spring Harb. Protoc. 2009,
pdb.prot5264.
Knoepfler, P. S., Bergstrom, D. A., Uetsuki, T., Dac-Korytko, I., Sun, Y. H.,
Wright, W. E., Tapscott, S. J. and Kamps, M. P. (1999). A conserved motif Nterminal to the DNA-binding domains of myogenic bHLH transcription factors
mediates cooperative DNA binding with pbx-Meis1/Prep1. Nucleic Acids Res. 27,
3752-3761.
Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H. and
Tanaka, E. M. (2009). Cells keep a memory of their tissue origin during axolotl
limb regeneration. Nature 460, 60-65.
Maden, M. (1980). Intercalary regeneration in the amphibian limb and the rule of
distal transformation. J. Embryol. Exp. Morphol. 56, 201-209.
Mathew, S. J., Hansen, J. M., Merrell, A. J., Murphy, M. M., Lawson, J. A.,
Hutcheson, D. A., Hansen, M. S., Angus-Hill, M. and Kardon, G. (2011).
Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 138,
371-384.
Mchedlishvili, L., Epperlein, H. H., Telzerow, A. and Tanaka, E. M. (2007). A
clonal analysis of neural progenitors during axolotl spinal cord regeneration
reveals evidence for both spatially restricted and multipotent progenitors.
Development 134, 2083-2093.
Mchedlishvili, L., Mazurov, V., Grassme, K. S., Goehler, K., Robl, B., Tazaki,
A., Roensch, K., Duemmler, A. and Tanaka, E. M. (2012). Reconstitution of
the central and peripheral nervous system during salamander tail regeneration.
Proc. Natl. Acad. Sci. USA 109, E2258-E2266.
Mercader, N., Leonardo, E., Azpiazu, N., Serrano, A., Morata, G., Martínez,
C. and Torres, M. (1999). Conserved regulation of proximodistal limb axis
development by Meis1/Hth. Nature 402, 425-429.
Development 140 (3)
Mercader, N., Tanaka, E. M. and Torres, M. (2005). Proximodistal identity during
vertebrate limb regeneration is regulated by Meis homeodomain proteins.
Development 132, 4131-4142.
Mohun, T. J., Garrett, N. and Gurdon, J. B. (1986). Upstream sequences
required for tissue-specific activation of the cardiac actin gene in Xenopus laevis
embryos. EMBO J. 5, 3185-3193.
Nacu, E. and Tanaka, E. M. (2011). Limb regeneration: a new development?
Annu. Rev. Cell Dev. Biol. 27, 409-440.
Nacu, E., Knapp, D., Tanaka, E. M. and Epperlein, H. H. (2009). Axolotl
(Ambystoma mexicanum) embryonic transplantation methods. Cold Spring
Harb. Protoc. 2009, pdb.prot5265.
Pescitelli, M. J. and Stocum, D. L. (1980). The origin of skeletal structures
during intercalary regeneration of larval Ambystoma limbs. Dev. Biol. 79, 255275.
Roselló-Díez, A., Ros, M. A. and Torres, M. (2011). Diffusible signals, not
autonomous mechanisms, determine the main proximodistal limb subdivision.
Science 332, 1086-1088.
Roy, S. and Vijayraghavan, K. (1997). Homeotic genes and the regulation of
myoblast migration, fusion, and fibre-specific gene expression during adult
myogenesis in Drosophila. Development 124, 3333-3341.
Stocum, D. (1975). Regulation after proximal or distal transposition of limb
regeneration blastemas and determination of the proximal boundary of the
regenerate. Dev. Biol. 45, 112-136.
Walthall, J. C. and Ashley-Ross, M. A. (2006). Postcranial myology of the
California newt, Taricha torosa. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 288,
46-57.
Yamamoto, M. and Kuroiwa, A. (2003). Hoxa-11 and Hoxa-13 are involved in
repression of MyoD during limb muscle development. Dev. Growth Differ. 45,
485-498.
Yamamoto, M., Gotoh, Y., Tamura, K., Tanaka, M., Kawakami, A., Ide, H.
and Kuroiwa, A. (1998). Coordinated expression of Hoxa-11 and Hoxa-13
during limb muscle patterning. Development 125, 1325-1335.
Yost, C., Torres, M., Miller, J. R., Huang, E., Kimelman, D. and Moon, R. T.
(1996). The axis-inducing activity, stability, and subcellular distribution of betacatenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes
Dev. 10, 1443-1454.
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