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Full regeneration of the tribasal Polypterus fin

Article  in  Proceedings of the National Academy of Sciences · March 2012

DOI: 10.1073/pnas.1006619109 · Source: PubMed

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Full regeneration of the tribasal Polypterus fin
Rodrigo Cuervoa,b, Rocío Hernández-Martíneza, Jesús Chimal-Monroyc, Horacio Merchant-Lariosc,
and Luis Covarrubiasa,1
a
Department of Developmental Genetics and Molecular Physiology, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca,
Morelos, 62210, Mexico; bFacultad de Ciencias Biológicas y Agropecuarias, Universidad Veracruzana, Tuxpan, Veracruz, 92850, Mexico; and cDepartment of
Cell Biology and Physiology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, 04510 Mexico City, D.F., Mexico
Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved January 11, 2012 (received for review May 12, 2010)
Full limb regeneration is a property that seems to be restricted to
urodele amphibians. Here we found that Polypterus, the most basal
living ray-finned fish, regenerates its pectoral lobed fins with a re-
markable accuracy. Pectoral Polypterus fins are complex, formed by
a well-organized endoskeleton to which the exoskeleton rays are
connected. Regeneration initiates with the formation of a blastema
similar to that observed in regenerating amphibian limbs. Retinoic
acid induces dose-dependent phenotypes ranging from inhibition
of regeneration to apparent anterior–posterior duplications. As in
all developing tetrapod limbs and regenerating amphibian blas-
tema, Sonic hedgehog is expressed in the posterior mesenchyme
during fin regeneration. Hedgehog signaling plays a role in the
regeneration and patterning processes: an increase or reduction
of fin bony elements results when this signaling is activated or
disrupted, respectively. The tail fin also regenerates but, in contrast
with pectoral fins, regeneration can resume after release from the
arrest caused by hedgehog inhibition. A comparative analysis of fin
phenotypes obtained after retinoic acid treatment or altering the
hedgehog signaling levels during regeneration allowed us to as-
sign a limb tetrapod equivalent segment to Polypterus fin skeletal
structures, thus providing clues to the origin of the autopod. We
propose that appendage regeneration was a common property of
vertebrates during the fin to limb transition.
development | evolution | actinopterygians | bichir
P aired appendages (i.e., fins and limbs) are a common charac-
teristic of most members of the subphylum Vertebrata, which
includes chondrichthyans, actinopterygians, and sarcopterygians.
Chondrichthyans encompass very ancient vertebrates such as
sharks, and actinopterygians include all ray-finned fishes (e.g.,
teleosts), whereas the sarcopterygian class contains lobe-finned
fishes and tetrapods. Appendages are supported by a cartilaginous
endoskeleton that usually ossifies in actinopterygians and sarcop-
terygians. Appendages of different vertebrate species (extant and
extinct) have distinct patterns of endoskeletal elements. Although
the identity and homology of bony elements can be assigned with
confidence in many cases, uncertainties arise when comparisons
are performed between members of different classes or subclasses
(1). In fishes, distal to the endoskeleton, there is a dermal exten-
sion supported by ossified fin rays (i.e., lepidotrichia) that usually
comprises the entire web of the fin. It is known that the dermal
skeleton disappeared during the evolution from tetrapod-like
fishes to early tetrapods; however, the origin of the autopod, the
most distal part of a limb, is still an area of controversy.
Polypterus has several primitive ancestral as well as derived
features that historically have complicated its phylogenetic posi-
tioning and hence its classification. Polypterus shares several
morphological and developmental characteristics with extinct
early tetrapods and extant amphibians (e.g., paired lungs origi-
nating from the ventral foregut, external gills, the structure of the
gill arches, stapes bone, spiracular openings, and pituitary gland)
(2–4). In the description of its early development, J. S. Budgett
mentioned the egg segmentation, movements during gastrulation,
and neural fold development as being astoundingly frog-like (5).
Recent molecular analysis places Polypterus as the most basal
actinopterygian fish (6). The pectoral fin of polypteriformes and
www.pnas.org/cgi/doi/10.1073/pnas.1006619109
other basal actinopterygians (Acipenseriformes, Semionotiformes,
Amiiformes), as well as most Chondrichthyes and fossils of the
earliest gnathostomes, can form a tribasal structure (7, 8). The
endoskeleton of a tribasal fin is composed of three proximal
skeletal structures named propterygium (anterior), metapterygium
(posterior), and mesopterygium (middle). Different authors have
postulated that the tribasal fin represents the basal condition for
the gnathostome pectoral fin (1, 8).
The accuracy and reproducibility of amphibian limb re-
generation has astonished many scientists since its discovery more
than 200 y ago (9). After limb amputation and healing, a structure
known as the blastema forms. From this structure limb growth
DEVELOPMENTAL
begins and, after approximately 30 d, a complete limb with the
BIOLOGY
exact pattern and size of the original is regenerated. This re-
generation implies the formation of all bones along the proximo-
distal (PD) axis of the limb: humerus/femur (or stylopod, the most
proximal part of the limb), radio and ulna/tibia and fibula (or
zeugopod, the middle part of the limb), and carpals, metacarpals,
and digits/tarsals, metatarsals, and digits (or autopod, the most
distal part of the limb).
The plesiomorphic and derived characters found in Poly-
pteriformes make these fishes attractive subjects for performing
evolutionary and developmental comparisons. In the present
work, we evaluated the ability of the Polypterus fin to regenerate
upon a quasi-complete amputation. We observed that Polypterus
regenerates its fins with remarkable accuracy, only comparable
to the regeneration observed in amphibian urodeles. The effects
observed on fin patterning caused by increasing retinoic acid
(RA) or altering Sonic hedgehog (Shh) signaling during re-
generation supports similarities between the distal segments of
the tetrapod limb and Polypterus fin.
Results
Bone Structure of the Polypterus Pectoral Fin. On the basis of the
anatomical similarities between the larval fins of Polypterus and
Heptanchus sharks, Budgett concluded that the large bone of the
Polypterus fin corresponds to the metapterygium and the short
bone to the propterygium (5). Proximally, the propterygium and
metapterygium bones meet and articulate with a convex head
bone that protrudes from the scapulocoracoid, whereas distally
they articulate with small skeletal elements (Fig. 1A). The radials
form a serial of transverse-aligned rod-like bones (approximately
13.6 radials, n = 20) forming a common axis of flexion and ex-
tension (Fig. 1A); each radial articulates in a one-to-one manner
Author contributions: R.C. and L.C. designed research; R.C. and R.H.-M. performed research;
R.H.-M., J.C.-M., and H.M.-L. contributed new reagents/analytic tools; R.C., R.H.-M., and L.C.
analyzed data; and R.C. and L.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. HM190156).
1
To whom correspondence should be addressed. E-mail: covs@ibt.unam.mx.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1006619109/-/DCSupplemental.
PNAS Early Edition | 1 of 6

Fig. 1. Anatomy of the Polypterus fin. (A) Frontal view of alcian blue- and
alizarin red-stained right fin and pectoral girdle, indicating the main fin
skeletal components: cleithrum (cl), lepidotrichia (lp), mesopterygium (ms),
metapterygium (mt), propterygium (pr), radials (rd), and scapulocoracoid
(sc). (Scale bar, 5 mm.) Note the small bone element (*) that articulates to
each of the large bone elements metapterygium and propterygium. Seg-
ments that may be equivalent to the autopod and zeugopod of tetrapods
are also indicated (autopodial- and zeugopodial-like; Discussion). (B) Close-
up of A to visualize the distal radials (drd) attached to each radial bone el-
ement. Posterior is to the bottom in A and to the left in B.
with a distal radial (Fig. 1B). Distally, a fan shaped lepidotrichia
skeleton rims the fin lobule (Fig. 1A).
Regeneration of Pectoral Fins. We discovered that Polypterus lobed
fins have a remarkable capacity to regenerate upon amputation.
Proximal amputations were performed, and complete re-
generation occurred within 1 mo (Fig. 2 A and B). The stump
forms a blastema within 3 d (Fig. 2C), and after 4 d the epithelial
basal stratum seems to transform into a protruding fold (i.e., basal
apical epidermis) that rims the blastema in an anterior–posterior
(AP) direction (Fig. 2 C and D); this is similar in appearance to the
apical fold of developing zebrafish fins (10). Rapidly this apical
fold expands and bends toward the dorsal side and is invaded by
mesenchymatous cells (Fig. 2D), thereby giving rise to the fin fold
that will develop into the endoskeleton and dermal skeleton.
Cartilage differentiation is observed in the expected bone forma-
tion area as soon as 9 days postamputation (dpa), closely analo-
gous to what is observed during tetrapod limb regeneration (11).
Precartilage condensations can be seen in histological slices, and
the chondroblasts underneath can be distinguished by their sparse
arrangements and the extracellular matrix surrounding them,
whereas the apical mesenchymatous cells of the regenerate remain
undifferentiated (Fig. 2D and Fig. S1). The endoskeleton regen-
erates by recapitulation of the embryonic developmental mecha-
nism (2). A continuous cartilaginous plate forms, whose thickened
margin foreshadows the differentiation of long bones, whereas
radials forms by perforation and splitting of the cartilaginous plate
(Fig. 2B). This pattern of skeletal formation disagrees with the
model of branching and segmentation for the origin of limb bone
elements (12). Actually, several observations suggest that serial
branching is not the mechanism by which bone elements are
formed during limb development (13).
Role of RA in Pectoral Fin Regeneration. RA treatment during re-
generation resulted in fin phenotypes characterized by the loss of
distal structures. The severity of the effect depended on the stage
at which blastema were exposed to RA (Fig. 3 and Table 1).
Early treatments (3–6 dpa) with 100 μg/g drastically reduced the
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Fig. 2. Pectoral fin regeneration in P. ornatipinnis and fin endoskeleton re-
generation in P. senegalus. (A) External appearance of the same fin during the
regeneration process at sequential days after amputation. “d0” shows a fin
before amputation. Anterior is to the right, and dorsal to the top. (Scale bar,
8 mm.) (B) Alcian blue- and alizarin red-stained regenerates at different days
after amputation (see also Fig. S1). Anterior is to the left. (Scale bar, 2.5 mm.)
(C) External appearance of blastema and apical fold in a 4-day regenerate. (D)
Toluidine blue-stained longitudinal sections of regenerates at different days
after amputation. Lower: Areas within the squares (Upper), shown at higher
magnification. Arrowhead in the 4d high-magnification picture indicates the
apical fold, a structure formed by basal stratum cells. Ventral is to the right.
(Scale bars, 400 μm in Upper, 40 μm in Lower.) bs, basal stratum; cc, cartilage
condensation; cp, cartilaginous plate; eg, external gill.
endoskeleton elements (Fig. 3A), but proximo-distal fin dupli-
cations were never observed. This was unexpected, because
performing the same protocol on regenerating Axolotl limbs
generated proximo-distal limb duplications in 95% of regener-
ates, as previously reported (14). Late treatments (9 dpa) caused
a reduction in the number of radials and lepidotrichia (Fig. 3A
and Table 1). The effects also were dependent on the RA con-
centration used (Table 1). Interestingly, we were able to obtain
phenotypes resembling mild to strong duplications of fin bones
with doses of 75 μg/g (Fig. 3B and Fig. S2), although near-normal
fins were generated at a slightly lower dose (50 μg/g; Table 1).
Therefore, as seen during amphibian limb regeneration, fin re-
generation is also sensitive to elevated doses of RA, producing
different phenotypes in a dose- and time-dependent manner
(Discussion).
Role of Shh in Pectoral Fin Regeneration. Data reported recently
demonstrate a correlation between the morphological diversity
of limbs and fins and the evolutionary changes in the expression
and regulation of the Sonic hedgehog gene (Shh) (15, 16). We
detected Shh expression in the posterior region of regenerating
Polypterus fins that extends and fades anteriorly below the
regenerating fin fold (Fig. 4A and Fig. S3), which is similar to the
expression pattern observed in fin buds of different fish embryos
(16) or regenerating urodele limbs (17). To determine the role of
Shh in fin regeneration and patterning, regenerates were exposed
to cyclopamine, an inhibitor of Shh signaling, or Hh-Ag1.3,
a hedgehog agonist. Treatment with cyclopamine (2 μg/mL) at
early stages (1–6 dpa) impaired regeneration (Fig. 4B). All fins
showed a severe reduction in the number of radials and lep-
idotrichia (20 of 20; Fig. 4 C and E); the most-affected fins
showed only one skeletal element and no lepidotrochia (Fig. 4C,
upper fin). Treatment at late stages (9–12 dpa) with a lower dose
(1 μg/mL) resulted in narrow fins with reduced number of radials
(19 of 19; Fig. 4 D and E). Maintaining a fixed concentration of
cyclopamine (2 μg/mL) from different regeneration stages on fins
of Polypterus ornatipinnis, we obtained stage-dependent sequen-
tial radial reduction phenotypes (Fig. 4H). Regeneration upon
Cuervo et al.

Fig. 3. Fin regenerates treated with RA. (A) Ventral view of right fin
regenerates treated with 100 μg/g RA at 3, 6, and 9 dpa, as described in
Materials and Methods. (B) Ventral view of a right fin treated with 75 μg/g
RA at 3 dpa, showing a phenotype resembling AP duplications; arrowhead
indicates a bend near an incipient second point of duplication. Right: En-
larged area shows a different view of the putative second duplication; note
the small lepidotrichia (arrowheads) shared by the adjacent fin lobe and
flanking small lobule. (C) Ventral view of a left fin treated with 75 μg/g RA at
3 dpa showing what appears to be the emergence of a large cartilage,
possibly corresponding to a duplicated propterigium or metapterigium;
arrowheads indicate lepidotrichia emerging from the new large cartilage.
Right: Enlarged area shows a dorsal view of the same fin. Arrows indicate
the point of main duplication. All fins shown were dissected and stained
with alcian blue and alizarin red 35 d after RA treatment.
tail fin lepidotrichia amputation was also blocked by cyclopamine
(Fig. 5 A and B) but, in contrast with endoskeleton regeneration,
it was resumed after cyclopamine removal (Fig. 5D).
On the other hand, treatments with Hh-Ag1.3 from early
stages (2 dpa) of fin regeneration produced dose-dependent
phenotypes characterized by broader fins with increased number
of radials and lepidotrichia (100 nM, Fig. 4 E and F; 200 nM, Fig.
4G). It is worth mentioning that, despite the consistent increase
in the number of radials with this Shh agonist, we never observed
phenotypes resembling AP duplications. The Shh agonist did
cause an effect on tail fin lepidotrichia regeneration, but their
number appeared unchanged (Fig. 5 A and C).
Discussion
Limb and Fin Regeneration. Regeneration of limbs of some uro-
deles has attracted a lot of attention because of its astonishing
precision, only comparable to the development of an organism.
Patterning, cellular components, and size are perfectly regulated,
such that a fully functional limb develops. Relevant advances
regarding the development of the regenerated limb have been
reported; however, the basis of why certain species are able to
regenerate their limbs is still unknown. Relevant questions to
answer are why/how this property was gained and why/how it was
lost. The ability to regenerate appendages does not seem to be
a property of early members of the vertebrate lineage because the
paired fins of sharks are unable to regenerate (18). As shown
here, Polypterus regenerates its pectoral lobed fins with remark-
able accuracy, reminiscent of limb regeneration in urodele
amphibians, fulfilling the concept of epimorphic regeneration.
Even though we used small fishes for most of the experiments
described, Polypterus is able to rebuild the complex structure of its
lobed fins at sizes approximately 35 cm long (close to the re-
productive stage), when fins are larger than an adult Ambystoma
autopod and the lobulated section is at least 2 cm wide. To our
Cuervo et al.
Table 1. Proportion of fin regenerates with a particular
phenotype after i.p. injection of RA (μg/g of body weight)
From 3 dpa
From 6 dpa From 9 dpa
Parameter 100 75 50 100 100
Normal 0/20 0/20 12/14 0/22 0/22
Shortened 14/20 12/20 0/14 15/22 12/22
AP duplication 0/20 4/20 0/14 0/22 0/22
PD duplication 0/20 0/20 0/14 0/22 0/22
Nonregenerated 6/20 4/20 2/14 5/22 2/22
Broadly misshapen 0/20 0/20 0/14 2/22 8/22
PD, proximo-distal; AP, anterior-posterior.
knowledge, African lungfishes, the closest living relatives of tet-
rapods, seem to be the only other vertebrate group in which re-
generation of the fin bony endoskeleton has been reported (19).
Although it is difficult to generalize owing to the great variety
of extant species, it is possible that the appendage regenerative
capacity decreased within different branches during the course of
vertebrate diversification. In teleost fishes, the ability to re-
generate is restricted to the dermal exoskeleton because fin
DEVELOPMENTAL
amputation close to the body has failed to show regeneration
BIOLOGY
(18). Besides urodeles, among terrestrial vertebrates only limited
regeneration has been observed in other amphibians such as
Xenopus laevis, particularly during larval stages. Mammals can-
not regenerate full limbs, although remnants of regeneration can
be observed at the tip of digits (20).
Despite a recent report suggesting that endogenous RA is not
involved in limb patterning in mouse and zebrafish (21), it is well
established that exogenous RA affects limb development and
regeneration (14, 22). In particular, during mouse, chick, and
axolotl development, exogenous RA causes stage-dependent
distal truncations, with the zeugopod and autopod the skeletal
elements being most affected (23–25). In contrast, during newt
and axolotl limb regeneration, exogenous RA induces proximo-
distal duplications (14, 25). In addition, in anurans such as X.
laevis, AP duplications of regenerated limb have been reported
(26). Limbs with mirror-image duplications are obtained in the
chick when an RA-soaked bead is implanted in their anterior
region during limb development. AP duplications primarily af-
fect bone elements of the autopod, but it is not uncommon to
also observe zeugopod elements duplicated. It was surprising to
find that the regenerating Polypterus fin behaved similar to de-
veloping and regenerating tetrapod limbs in response to exoge-
nous RA. In particular, distal fin truncations and phenotypes
resembling AP duplications appeared equivalent to the autopod
phenotype of developing limbs treated with RA. Despite this
similarity, mechanistic differences may exist between Polypterus
and amphibian regeneration: we did not observe proximo-distal
duplication under any of the RA treatment conditions used in
this work, and to our knowledge, AP duplications have not been
observed in regenerating urodele limbs. In the developing limb
(as may occur in the Polypterus fin), RA kills the prespecified
distal mesenchyme (27) and/or acts as a proximilizing factor on
the distal mesenchyme (23–25), whereas in the regenerating
amphibian limb, RA seems to reset the mesenchyme to the most
proximal identity regardless of the stage of treatment (23–25).
Members of the hedgehog family have key functions in the de-
velopment of all animals. In the limb of vertebrates, Shh is the
essential component of the zone of polarizing activity. The phe-
notype of mouse limbs carrying null Shh alleles shows distal ab-
normalities, characterized by a zeugopod with a single bone,
possibly the radius, and a reduced number of digits, leaving what
seems to be digit one (28, 29). Similarly, cyclopamine induces
a posterior to anterior digit loss in a dose-dependent fashion in
PNAS Early Edition | 3 of 6

Fig. 4. Fin regenerates treated with cyclopamine and Shh agonist. (A)
Frontal view of a 10 dpa right fin regenerate hybridized with an Shh probe
(same position as Fig. 1A). Arrows point to the cleithrum bone and scapula.
(B) Ventral and dorsal view of control (Top and Bottom Left, respectively)
and cyclopamine-treated (Middle and Bottom Right, respectively) pectoral
fins after 35 d of regeneration. (C) Skeletal staining of early regenerates
treated with 2 μg/mL cyclopamine (Cyc). Right: Magnifications of fins shown
at Left. Arrowhead marks the limit of the anterior (to the right) region af-
fected. (D) Skeletal staining of late regenerates treated with 1 μg/mL
cyclopamine. (E) Skeletal staining of an early regenerate treated with DMSO
(Control). (F) Skeletal staining of regenerates treated with 100 nM Hh-Ag1.3.
(G) Skeletal staining of a regenerate treated with 200 nM Hh-Ag1.3. Ar-
rowhead marks the AP limit at which the posterior region appears more
affected. (H) Skeletal staining of left fin regenerates and pectoral girdles of
P. ornatipinnis after treatment with 2 μg/mL cyclopmine at 8, 10, 12, and 14
dpa and then left regenerating for 3 wk; control is an untreated fin
regenerated for 42 d. Note the sequential reduction of the mesopterygial
plate and lepidotrichia, with minor disturbances of propterygium and met-
apterygium bones. Materials and Methods provides details about treatment
protocols. a, anterior; cl, cleithrum; d, dorsal; p, posterior; sc, scapulocora-
coid; v, ventral.
regenerating axolotl limbs (15, 30), phenocopying the autopod
abnormalities observed in the Shh mutant mice. On the other
hand, ectopic Shh added to the anterior region of developing chick
limbs produces extra digits with a mirror digit identity (31), and
ectopic expression of Shh (32) or treatment with a Smoothened
agonist in axolotl regenerating limbs does not induce AP dupli-
cations but causes extra digits, carpals, and tarsals to form. In ad-
dition, lack of Gli3, a fundamental target of Shh signaling in the
limb, produces polydactyly (28). From these data, it has been
concluded that Shh regulates the patterning and number of distal
bone elements of the limb, starting from the zeugopod. In Poly-
pterus, inhibiting or activating Shh signaling during fin regeneration
affected the number of radials, in parallel with the effects observed
in the number of digits in terrestrial vertebrates, including the
posterior–anterior responsiveness to Shh dose.
Homology Between Polypterus Pectoral Fin and Tetrapod Limbs: A
Proposal with Evolutive Implications. The detailed comparison of
Polypterus fin bone elements with radius/radiale, ulna/ulnare,
and intermedium, the mesopodial most conserved elements in
tetrapod-like fishes and extinct and extant tetrapods, is difficult
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Fig. 5. Effects of altering Shh signaling on regeneration of fin tail lepido-
trichia. (A) Alizarin red staining of control (DMSO-treated) tail fin. (B) Tail fin
treated with 2 μg/mL cyclopamine. (C) Tail fin treated with 100 μM of Shh
agonist. (D) Regeneration of tail fin before and 3 wk after cyclopamine
withdrawal. Fin pictures were taken at 3 wk after amputation, except for the
picture in D. Cyclopamine impairs the regenerative process (A and B), but it is
resumed after cyclopamine withdrawal (D). The Hh-Ag3.1 agonist did not
affect the number, but the morphology of the regenerating tail fin lep-
idotrichia showed a zigzag pattern (C).
to address without bias. According to the bone arrangement
described here, the large and small bone elements (metaptery-
gium and propterygium, respectively) may lie at an equivalent
segment as the tetrapod radius/ulna and radiale/ulnare (Fig. 1A);
the nodular bone observed between these long bones can be
homologized with the intermedium (Fig. 1A). In addition, the
articulation between the small skeletal elements (radials) and
the propterygium and metapterygium, forming a common axis of
flexion and extension, might constitute a “primordial wrist.” In
several early tetrapods, wrist bones are almost absent, and it
seems that the evolution of ankle and wrist joints lagged behind
the formation of digits (1). Currently, the presence of more than
five digits (hence, metacarpals) is accepted as a plesiomorphic
character of the autopod (33); in Polypterus, approximately 12–
14 radials are commonly found. In concordance with an equiv-
alence between the distal Polypterus fin and the autopod (Fig.
1A), it has been noted that the only elements of the fin whose
development resembles that of the autopodial elements are the
distal radials, a cartilaginous structure similar in appearance to
distal phalanges (34).
On the basis of the similarities described above and the
observations resulting from altering RA and Shh signaling during
Polyperus fin regeneration, radials would seem equivalent to
metacarpals of the tetrapod autopod (see below), and the prop-
terygium and metapterygium seem equivalent to the zeugopod
bone elements (autopodial- and zeugopodial-like, respectively;
Fig. 1A). The homology proposed is in concordance with recent
data showing that treatment of skate embryos with RA provokes
the expression of Shh in the anterior region of fins and conse-
quently the propterygium acquires the identity of the metaptery-
gium (16). This change in identity resembles the transformation of
radius to ulna of tetrapod limbs treated with RA, Shh, or through
transplantation of the polarizing zone (31, 35, 36).
A corollary of the equivalence of radials to metacarpals is that
the autopod is an ancient structure that existed before the
emergence of tetrapods. This contrasts with a commonly accepted
view in which the autopod is considered a structure appearing de
novo in tetrapods. The proposed primitive origin of the autopod is
in agreement with the autopodial-like Hox expression pattern
Cuervo et al.
recently determined in Polyodon spathula fin, another basal acti-
nopterygian fish (37), and in Scyliorhinus canicula, a member of
the most basal lineage of jawed vertebrates (38). Also, Hoxd13
expression in the Australian lungfish closely matches the late
expression patterns observed in the tetrapod autopod (39). These
data together suggest that fin radials and tetrapod digits may be
patterned by shared mechanisms that are distinct from those
patterning the proximal fin/limb bone elements. This gene ex-
pression pattern homology has led to the deep homology concept
in which a specific gene regulation mechanism is used to build
structures with no evident phylogenetic or morphologic re-
lationship (40). Thus, shared gene expression patterns may not
necessarily indicate structural homology or evolutionary origin
(41). In the present report we observed that comparative analyses
of the RA and Shh responses provide additional clues to un-
derstand appendicular skeleton evolution. Thus, in terms of deep
homology, we must consider, in addition to shared genetic pro-
grams, shared responses of developing/regenerating structures to
morphogens. The remarkable ability of Polypterus to regenerate
their pectoral fins will allow a detailed comparative analysis of
gene expression patterns in combination with growth factor
responses and, in this manner, to experimentally test whether the
tribasal structure of Polypterus is the ancestor of the tetrapod
zeugopod and autopod.
Materials and Methods
Whole-Mount Skeletal Preparations. Commercially obtained juvenile bichirs,
Polypterus senegalus and P. ornatipinnis, ≈6 cm long shortly after gill re-
duction phase (2), were used for the experiments. Fishes were maintained in
glass tanks at 28 °C. Amputations were performed with nail clippers after
anesthesia by immersion in cold water containing a 0.1% tricaine solution.
Amputations were performed closer to the body. Bichirs were killed with an
overdose of tricaine; the fins were dissected, fixed and, dehydrated overnight
(ON) in 100% ethanol. Fins were incubated in acetone for 24 h and then
stained for 3 h at 37 °C plus ON at room temperature in alcian blue and alizarin
red in 70% ethanol with 5% acetic acid. Fins were rinsed in tap water before
clearing in 1% KOH and 20% glycerol for 24 h and then in graded glycerol.
Histology. Regenerates were fixed in modified Karnovsky for 5 h at 4 °C and
then washed ON in sodium cacodylate buffer at 4 °C. After a postfixation in
1% osmium, samples were dehydrated in graded ethanol series and em-
bedded in Epon. Half-micrometer sections were stained with toluidine blue
and visualized by bright-field microscopy.
Whole-Mount in Situ Hybirdization. Fins were fixed ON with MEMPFA [0.1 M
Mops (pH 7.4), 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde]. The in situ
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hybridization protocol used was based on that described by Nieto et al. (42),
with two major modifications: proteinase K treatment was extended up to
45 min for large pieces comprising the cleithrum/scapulocoracoid bones, and
the last wash in TBST [for 1 L of 10× TBST: 8 g NaCl, 0.2 g KCl, 25 mL of 1 M
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RA, Cyclopamine, and Shh Agonist Treatment. Bichirs were transferred on small
glass containers with 100 mL of water. Cyclopamine (BIOMOL) was adminis-
trated by adding it directly to water at 2 μg/mL or 1 μg/mL final concentration
from a 5 mg/mL stock in DMSO. Hh-Ag1.3 (Curis) was used at 100 and 200 nM
DEVELOPMENTAL
from a 1-mM stock in DMSO. Treatments with a commercially available
BIOLOGY
Smoothened agonist SAG (Calbiochem) at 200 nM final concentration gave
similar effects. Equivalent amounts of DMSO were added to controls. Treat-
ments were performed for 3 wk starting at 1–9 d (early stages) or 12–18 d
(late stages) after amputation. Water containing cyclopamine or Hh-Ag1.3
was changed every day during treatment. After treatment, regeneration was
allowed to continue for a further 3 wk, before fishes were killed and fins
dissected.
The protocol for RA injection was as reported for urodeles (14). All-trans-RA
(Sigma) dissolved in DMSO (15 mg/mL) was injected i.p. (100, 75, or 50 μg/g of
body weight), on day 3 after amputation. For time-dependant studies, sub-
sequent injections were done at 6 and 9 d after amputation with 100 μg/g of
body weight.
ACKNOWLEDGMENTS. We thank Dr. Chris Wood for discussion of data and
careful reading of the manuscript; Marcia Bustamante, Concepción Valencia,
and Sr. José G. Baltazar García for their technical assistance; and Sr. Dionisio
Eslava of the biological station Umbral Axochiatl A.C. for supplying the
axolotl larvae. Hh-Ag1.3 was donated by Curis, Inc. This work was supported
by Grant IN218607 from Programa de Apoyo a Proyectos de Investigación e
Innovación Tecnológica-Dirección General Asuntos del Personal Académico-
Universidad Nacional Autónoma de México (PAPIIT-DGAPA) and Grant
39930-Q from Consejo Nacional de Ciencia y Tecnología (CONACyT).
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Supporting Information
Cuervo et al. 10.1073/pnas.1006619109

Fig. S1. Chondrogenesis at 9 d postamputation (dpa). (A) Alcian blue- and alizarin red stained right fin at 9 dpa. Arrow indicates the limit between the
chondrogenic endoskeleton (presumptive radials) and exoskeleton (presumptive lepidotrichia). (B) High magnification of a distal area of sample shown in A,
showing the avascular finfold region (upper and lower borders delimited by the vertical line) and the subjacent vascularized region positive for alcian blue
staining; blood vessels are conspicuously seen within the blue background. (C) Toluidine blue-stained section of the proximal third of a regenerate at 9 dpa;
chondroblasts are distinguished by their sparse arrangements and the abundant extracellular matrix surrounding them. (D) Distal region of the same regenerate;
arrow indicates the border between the apical mesenchymatous undifferentiated cells and the condensing cells of the prechondrogenic area. (Scale bar, 50 μm.)

Fig. S2. Phenotype of fins treated with intermediate doses of retinoic acid. Regenerating fins were treated with 75 μg/g of body weight at 3 d post-
amputation (dpa). At 35 dpa the fins were dissected and stained with alcian blue to visualize the skeletal elements regenerated. At a different degree, the two
samples show phenotypes resembling partial anterior–posterior duplications. Arrows indicate the apparent point of duplication.

Cuervo et al. www.pnas.org/cgi/content/short/1006619109 1 of 2

Fig. S3. Detection of Shh expression in Polyperus fin by whole-mount in situ hybridization. Different fins hybridized against a Polypterus antisense Shh probe
(AS) are shown. (A and B) Two fins hybridized against the sense probe (S) show no signal (Left), whereas fins at a similar regeneration stage hybridized with the
AS probe show Shh expression in the posterior region extending and fading anteriorly below the regenerating fin fold (Right). (C) Shh expression pattern in
fins at different regeneration stages.

Cuervo et al. www.pnas.org/cgi/content/short/1006619109 2 of 2

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