Full Regeneration of the Tribasal Polypterus ﬁn

Full regeneration of the tribasal Polypterus ﬁn

Rodrigo Cuervoa,b, Rocío Hernández-Martíneza, Jesús Chimal-Monroyc, Horacio Merchant-Lariosc, and Luis Covarrubiasa,1

aDepartment 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 other basal actinopterygians (Acipenseriformes, Semionotiformes, urodele amphibians. Here we found that Polypterus, the most basal Amiiformes), as well as most Chondrichthyes and fossils of the living ray-finned fish, regenerates its pectoral lobed fins with a re- earliest gnathostomes, can form a tribasal structure (7, 8). The markable accuracy. Pectoral Polypterus fins are complex, formed by endoskeleton of a tribasal fin is composed of three proximal a well-organized endoskeleton to which the exoskeleton rays are skeletal structures named propterygium (anterior), metapterygium connected. Regeneration initiates with the formation of a blastema (posterior), and mesopterygium (middle). Different authors have similar to that observed in regenerating amphibian limbs. Retinoic postulated that the tribasal fin represents the basal condition for acid induces dose-dependent phenotypes ranging from inhibition the gnathostome pectoral fin (1, 8). – of regeneration to apparent anterior posterior duplications. As in The accuracy and reproducibility of amphibian limb re- all developing tetrapod limbs and regenerating amphibian blas- generation has astonished many scientists since its discovery more tema, Sonic hedgehog is expressed in the posterior mesenchyme fi than 200 y ago (9). After limb amputation and healing, a structure during n regeneration. Hedgehog signaling plays a role in the known as the blastema forms. From this structure limb growth regeneration and patterning processes: an increase or reduction begins and, after approximately 30 d, a complete limb with the of fin bony elements results when this signaling is activated or exact pattern and size of the original is regenerated. This re- disrupted, respectively. The tail fin also regenerates but, in contrast generation implies the formation of all bones along the proximo- with pectoral fins, regeneration can resume after release from the fi distal (PD) axis of the limb: humerus/femur (or stylopod, the most arrest caused by hedgehog inhibition. A comparative analysis of n fi phenotypes obtained after retinoic acid treatment or altering the proximal part of the limb), radio and ulna/tibia and bula (or hedgehog signaling levels during regeneration allowed us to as- zeugopod, the middle part of the limb), and carpals, metacarpals, sign a limb tetrapod equivalent segment to Polypterus fin skeletal and digits/tarsals, metatarsals, and digits (or autopod, the most structures, thus providing clues to the origin of the autopod. We distal part of the limb). propose that appendage regeneration was a common property of The plesiomorphic and derived characters found in Poly- vertebrates during the fin to limb transition. pteriformes make these fishes attractive subjects for performing evolutionary and developmental comparisons. In the present development | evolution | actinopterygians | bichir work, we evaluated the ability of the Polypterus fin to regenerate upon a quasi-complete amputation. We observed that Polypterus fi aired appendages (i.e., fins and limbs) are a common charac- regenerates its ns with remarkable accuracy, only comparable Pteristic of most members of the subphylum Vertebrata, which to the regeneration observed in amphibian urodeles. The effects includes chondrichthyans, actinopterygians, and sarcopterygians. observed on fin patterning caused by increasing retinoic acid Chondrichthyans encompass very ancient vertebrates such as (RA) or altering Sonic hedgehog (Shh) signaling during re- sharks, and actinopterygians include all ray-finned fishes (e.g., generation supports similarities between the distal segments of teleosts), whereas the sarcopterygian class contains lobe-finned the tetrapod limb and Polypterus fin. fishes and tetrapods. Appendages are supported by a cartilaginous endoskeleton that usually ossifies in actinopterygians and sarcop- Results terygians. Appendages of different vertebrate species (extant and Bone Structure of the Polypterus Pectoral Fin. On the basis of the extinct) have distinct patterns of endoskeletal elements. Although anatomical similarities between the larval fins of Polypterus and the identity and homology of bony elements can be assigned with Heptanchus sharks, Budgett concluded that the large bone of the confidence in many cases, uncertainties arise when comparisons Polypterus fin corresponds to the metapterygium and the short are performed between members of different classes or subclasses bone to the propterygium (5). Proximally, the propterygium and (1). In fishes, distal to the endoskeleton, there is a dermal exten- metapterygium bones meet and articulate with a convex head sion supported by ossified fin rays (i.e., lepidotrichia) that usually bone that protrudes from the scapulocoracoid, whereas distally comprises the entire web of the fin. It is known that the dermal they articulate with small skeletal elements (Fig. 1A). The radials skeleton disappeared during the evolution from tetrapod-like form a serial of transverse-aligned rod-like bones (approximately fi shes to early tetrapods; however, the origin of the autopod, the 13.6 radials, n = 20) forming a common axis of flexion and ex- most distal part of a limb, is still an area of controversy. tension (Fig. 1A); each radial articulates in a one-to-one manner Polypterus has several primitive ancestral as well as derived features that historically have complicated its phylogenetic posi- fi Polypterus tioning and hence its classi cation. shares several Author contributions: R.C. and L.C. designed research; R.C. and R.H.-M. performed research; morphological and developmental characteristics with extinct R.H.-M., J.C.-M., and H.M.-L. contributed new reagents/analytic tools; R.C., R.H.-M., and L.C. early tetrapods and extant amphibians (e.g., paired lungs origi- analyzed data; and R.C. and L.C. wrote the paper. nating from the ventral foregut, external gills, the structure of the The authors declare no conflict of interest. gill arches, stapes bone, spiracular openings, and pituitary gland) This article is a PNAS Direct Submission. – (2 4). In the description of its early development, J. S. Budgett Data deposition: The sequence reported in this paper has been deposited in the GenBank mentioned the egg segmentation, movements during gastrulation, database (accession no. HM190156). and neural fold development as being astoundingly frog-like (5). 1To whom correspondence should be addressed. E-mail: [email protected]. Polypterus Recent molecular analysis places as the most basal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. actinopterygian fish (6). The pectoral fin of polypteriformes and 1073/pnas.1006619109/-/DCSupplemental.

3838–3843 | PNAS | March 6, 2012 | vol. 109 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1006619109 Downloaded by guest on October 1, 2021 Fig. 2. Pectoral fin regeneration in P. ornatipinnis and fin endoskeleton regeneration in P. senegalus.(A) External appearance of the same fin during the regeneration process at sequential days after amputation. “d0” shows a fin Fig. 1. Anatomy of the Polypterus fin. (A) Frontal view of alcian blue- and before amputation. Anterior is to the right, and dorsal to the top. (Scale bar, alizarin red-stained right fin and pectoral girdle, indicating the main fin 8 mm.) (B) Alcian blue- and alizarin red-stained regenerates at different days skeletal components: cleithrum (cl), lepidotrichia (lp), mesopterygium (ms), after amputation (see also Fig. S1). Anterior is to the left. (Scale bar, 2.5 mm.) metapterygium (mt), propterygium (pr), radials (rd), and scapulocoracoid (C) External appearance of blastema and apical fold in a 4-day regenerate. (D) (sc). (Scale bar, 5 mm.) Note the small bone element (*) that articulates to Toluidine blue-stained longitudinal sections of regenerates at different days each of the large bone elements metapterygium and propterygium. Seg- after amputation. Lower: Areas within the squares (Upper), shown at higher ments that may be equivalent to the autopod and zeugopod of tetrapods magnification. Arrowhead in the 4d high-magnification picture indicates the are also indicated (autopodial- and zeugopodial-like; Discussion). (B) Close- apical fold, a structure formed by basal stratum cells. Ventral is to the right. up of A to visualize the distal radials (drd) attached to each radial bone el- (Scale bars, 400 μminUpper,40μminLower.) bs, basal stratum; cc, cartilage ement. Posterior is to the bottom in A and to the left in B. condensation; cp, cartilaginous plate; eg, external gill.

with a distal radial (Fig. 1B). Distally, a fan shaped lepidotrichia endoskeleton elements (Fig. 3A), but proximo-distal fin dupli- skeleton rims the fin lobule (Fig. 1A). cations were never observed. This was unexpected, because performing the same protocol on regenerating Axolotl limbs Regeneration of Pectoral Fins. We discovered that Polypterus lobed generated proximo-distal limb duplications in 95% of regener- fins have a remarkable capacity to regenerate upon amputation. ates, as previously reported (14). Late treatments (9 dpa) caused Proximal amputations were performed, and complete re- a reduction in the number of radials and lepidotrichia (Fig. 3A generation occurred within 1 mo (Fig. 2 A and B). The stump and Table 1). The effects also were dependent on the RA con- forms a blastema within 3 d (Fig. 2C), and after 4 d the epithelial centration used (Table 1). Interestingly, we were able to obtain basal stratum seems to transform into a protruding fold (i.e., basal phenotypes resembling mild to strong duplications of fin bones apical epidermis) that rims the blastema in an anterior–posterior with doses of 75 μg/g (Fig. 3B and Fig. S2), although near-normal (AP) direction (Fig. 2 C and D); this is similar in appearance to the fins were generated at a slightly lower dose (50 μg/g; Table 1). apical fold of developing zebrafish fins (10). Rapidly this apical Therefore, as seen during amphibian limb regeneration, fin re- fold expands and bends toward the dorsal side and is invaded by generation is also sensitive to elevated doses of RA, producing mesenchymatous cells (Fig. 2D), thereby giving rise to the fin fold different phenotypes in a dose- and time-dependent manner that will develop into the endoskeleton and dermal skeleton. (Discussion). Cartilage differentiation is observed in the expected bone formation area as soon as 9 days postamputation (dpa), closely analo- Role of Shh in Pectoral Fin Regeneration. Data reported recently gous to what is observed during tetrapod limb regeneration (11). demonstrate a correlation between the morphological diversity Precartilage condensations can be seen in histological slices, and of limbs and fins and the evolutionary changes in the expression the chondroblasts underneath can be distinguished by their sparse and regulation of the Sonic hedgehog gene (Shh) (15, 16). We arrangements and the extracellular matrix surrounding them, detected Shh expression in the posterior region of regenerating whereas the apical mesenchymatous cells of the regenerate remain Polypterus fins that extends and fades anteriorly below the undifferentiated (Fig. 2D and Fig. S1). The endoskeleton regen- regenerating fin fold (Fig. 4A and Fig. S3), which is similar to the erates by recapitulation of the embryonic developmental mecha- expression pattern observed in fin buds of different fish embryos nism (2). A continuous cartilaginous plate forms, whose thickened (16) or regenerating urodele limbs (17). To determine the role of margin foreshadows the differentiation of long bones, whereas Shh in fin regeneration and patterning, regenerates were exposed radials forms by perforation and splitting of the cartilaginous plate to cyclopamine, an inhibitor of Shh signaling, or Hh-Ag1.3, (Fig. 2B). This pattern of skeletal formation disagrees with the a hedgehog agonist. Treatment with cyclopamine (2 μg/mL) at model of branching and segmentation for the origin of limb bone early stages (1–6 dpa) impaired regeneration (Fig. 4B). All fins elements (12). Actually, several observations suggest that serial showed a severe reduction in the number of radials and lep- branching is not the mechanism by which bone elements are idotrichia (20 of 20; Fig. 4 C and E); the most-affected fins formed during limb development (13). showed only one skeletal element and no lepidotrochia (Fig. 4C, upper fin). Treatment at late stages (9–12 dpa) with a lower dose Role of RA in Pectoral Fin Regeneration. RA treatment during re- (1 μg/mL) resulted in narrow fins with reduced number of radials generation resulted in fin phenotypes characterized by the loss of (19 of 19; Fig. 4 D and E). Maintaining a fixed concentration of

distal structures. The severity of the effect depended on the stage cyclopamine (2 μg/mL) from different regeneration stages on ﬁns BIOLOGY Polypterus ornatipinnis

at which blastema were exposed to RA (Fig. 3 and Table 1). of , we obtained stage-dependent sequen- DEVELOPMENTAL Early treatments (3–6 dpa) with 100 μg/g drastically reduced the tial radial reduction phenotypes (Fig. 4H). Regeneration upon

Cuervo et al. PNAS | March 6, 2012 | vol. 109 | no. 10 | 3839 Downloaded by guest on October 1, 2021 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 tetrapods, seem to be the only other vertebrate group in which regeneration of the fin bony endoskeleton has been reported (19). Although it is difficult to generalize owing to the great variety Fig. 3. Fin regenerates treated with RA. (A) Ventral view of right fin of extant species, it is possible that the appendage regenerative μ regenerates treated with 100 g/g RA at 3, 6, and 9 dpa, as described in capacity decreased within different branches during the course of Materials and Methods.(B) Ventral view of a right fin treated with 75 μg/g vertebrate diversification. In teleost fishes, the ability to re- RA at 3 dpa, showing a phenotype resembling AP duplications; arrowhead fi indicates a bend near an incipient second point of duplication. Right: En- generate is restricted to the dermal exoskeleton because n larged area shows a different view of the putative second duplication; note amputation close to the body has failed to show regeneration the small lepidotrichia (arrowheads) shared by the adjacent fin lobe and (18). Besides urodeles, among terrestrial vertebrates only limited flanking small lobule. (C) Ventral view of a left fin treated with 75 μg/g RA at regeneration has been observed in other amphibians such as 3 dpa showing what appears to be the emergence of a large cartilage, Xenopus laevis, particularly during larval stages. Mammals can- possibly corresponding to a duplicated propterigium or metapterigium; not regenerate full limbs, although remnants of regeneration can arrowheads indicate lepidotrichia emerging from the new large cartilage. be observed at the tip of digits (20). Right: Enlarged area shows a dorsal view of the same fin. Arrows indicate fi Despite a recent report suggesting that endogenous RA is not the point of main duplication. All ns shown were dissected and stained fi with alcian blue and alizarin red 35 d after RA treatment. involved in limb patterning in mouse and zebra sh (21), it is well established that exogenous RA affects limb development and regeneration (14, 22). In particular, during mouse, chick, and tail fin lepidotrichia amputation was also blocked by cyclopamine axolotl development, exogenous RA causes stage-dependent (Fig. 5 A and B) but, in contrast with endoskeleton regeneration, distal truncations, with the zeugopod and autopod the skeletal it was resumed after cyclopamine removal (Fig. 5D). elements being most affected (23–25). In contrast, during newt On the other hand, treatments with Hh-Ag1.3 from early and axolotl limb regeneration, exogenous RA induces proximo- stages (2 dpa) of fin regeneration produced dose-dependent distal duplications (14, 25). In addition, in anurans such as X. phenotypes characterized by broader fins with increased number laevis, AP duplications of regenerated limb have been reported of radials and lepidotrichia (100 nM, Fig. 4 E and F; 200 nM, Fig. (26). Limbs with mirror-image duplications are obtained in the 4G). It is worth mentioning that, despite the consistent increase chick when an RA-soaked bead is implanted in their anterior in the number of radials with this Shh agonist, we never observed region during limb development. AP duplications primarily af- phenotypes resembling AP duplications. The Shh agonist did fect bone elements of the autopod, but it is not uncommon to cause an effect on tail fin lepidotrichia regeneration, but their also observe zeugopod elements duplicated. It was surprising to number appeared unchanged (Fig. 5 A and C). find that the regenerating Polypterus fin behaved similar to developing and regenerating tetrapod limbs in response to exoge- Discussion nous RA. In particular, distal fin truncations and phenotypes Limb and Fin Regeneration. Regeneration of limbs of some uro- resembling AP duplications appeared equivalent to the autopod deles has attracted a lot of attention because of its astonishing phenotype of developing limbs treated with RA. Despite this precision, only comparable to the development of an organism. similarity, mechanistic differences may exist between Polypterus Patterning, cellular components, and size are perfectly regulated, and amphibian regeneration: we did not observe proximo-distal such that a fully functional limb develops. Relevant advances duplication under any of the RA treatment conditions used in regarding the development of the regenerated limb have been this work, and to our knowledge, AP duplications have not been reported; however, the basis of why certain species are able to observed in regenerating urodele limbs. In the developing limb regenerate their limbs is still unknown. Relevant questions to (as may occur in the Polypterus fin), RA kills the prespecified answer are why/how this property was gained and why/how it was distal mesenchyme (27) and/or acts as a proximilizing factor on lost. The ability to regenerate appendages does not seem to be the distal mesenchyme (23–25), whereas in the regenerating a property of early members of the vertebrate lineage because the amphibian limb, RA seems to reset the mesenchyme to the most paired fins of sharks are unable to regenerate (18). As shown proximal identity regardless of the stage of treatment (23–25). here, Polypterus regenerates its pectoral lobed fins with remark- Members of the hedgehog family have key functions in the de- able accuracy, reminiscent of limb regeneration in urodele velopment of all animals. In the limb of vertebrates, Shh is the amphibians, fulfilling the concept of epimorphic regeneration. essential component of the zone of polarizing activity. The phe- Even though we used small fishes for most of the experiments notype of mouse limbs carrying null Shh alleles shows distal ab- described, Polypterus is able to rebuild the complex structure of its normalities, characterized by a zeugopod with a single bone, lobed fins at sizes approximately 35 cm long (close to the re- possibly the radius, and a reduced number of digits, leaving what productive stage), when fins are larger than an adult Ambystoma seems to be digit one (28, 29). Similarly, cyclopamine induces autopod and the lobulated section is at least 2 cm wide. To our a posterior to anterior digit loss in a dose-dependent fashion in

3840 | www.pnas.org/cgi/doi/10.1073/pnas.1006619109 Cuervo et al. Downloaded by guest on October 1, 2021 Fig. 5. Effects of altering Shh signaling on regeneration of fin tail lepidotrichia. (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 μMofShh 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 Fig. 4. Fin regenerates treated with cyclopamine and Shh agonist. (A) affect the number, but the morphology of the regenerating tail fin lep- Frontal view of a 10 dpa right fin regenerate hybridized with an Shh probe idotrichia showed a zigzag pattern (C). (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 to address without bias. According to the bone arrangement fins after 35 d of regeneration. (C) Skeletal staining of early regenerates described here, the large and small bone elements (metaptery- treated with 2 μg/mL cyclopamine (Cyc). Right: Magnifications of fins shown gium and propterygium, respectively) may lie at an equivalent at Left. Arrowhead marks the limit of the anterior (to the right) region af- segment as the tetrapod radius/ulna and radiale/ulnare (Fig. 1A); μ fected. (D) Skeletal staining of late regenerates treated with 1 g/mL the nodular bone observed between these long bones can be cyclopamine. (E) Skeletal staining of an early regenerate treated with DMSO homologized with the intermedium (Fig. 1A). In addition, the (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- articulation between the small skeletal elements (radials) and rowhead marks the AP limit at which the posterior region appears more the propterygium and metapterygium, forming a common axis of affected. (H) Skeletal staining of left fin regenerates and pectoral girdles of flexion and extension, might constitute a “primordial wrist.” In P. ornatipinnis after treatment with 2 μg/mL cyclopmine at 8, 10, 12, and 14 several early tetrapods, wrist bones are almost absent, and it dpa and then left regenerating for 3 wk; control is an untreated fin seems that the evolution of ankle and wrist joints lagged behind regenerated for 42 d. Note the sequential reduction of the mesopterygial the formation of digits (1). Currently, the presence of more than plate and lepidotrichia, with minor disturbances of propterygium and met- five digits (hence, metacarpals) is accepted as a plesiomorphic apterygium bones. Materials and Methods provides details about treatment character of the autopod (33); in Polypterus,approximately12– protocols. a, anterior; cl, cleithrum; d, dorsal; p, posterior; sc, scapulocoracoid; v, ventral. 14 radials are commonly found. In concordance with an equivalence between the distal Polypterus fin and the autopod (Fig. 1A), it has been noted that the only elements of the finwhose regenerating axolotl limbs (15, 30), phenocopying the autopod development resembles that of the autopodial elements are the abnormalities observed in the Shh mutant mice. On the other distal radials, a cartilaginous structure similar in appearance to hand, ectopic Shh added to the anterior region of developing chick distal phalanges (34). limbs produces extra digits with a mirror digit identity (31), and On the basis of the similarities described above and the Shh observations resulting from altering RA and Shh signaling during ectopic expression of (32) or treatment with a Smoothened Polyperus fi agonist in axolotl regenerating limbs does not induce AP dupli- n regeneration, radials would seem equivalent to metacarpals of the tetrapod autopod (see below), and the prop- cations but causes extra digits, carpals, and tarsals to form. In ad- terygium and metapterygium seem equivalent to the zeugopod dition, lack of Gli3, a fundamental target of Shh signaling in the bone elements (autopodial- and zeugopodial-like, respectively; limb, produces polydactyly (28). From these data, it has been Fig. 1A). The homology proposed is in concordance with recent concluded that Shh regulates the patterning and number of distal data showing that treatment of skate embryos with RA provokes Poly- bone elements of the limb, starting from the zeugopod. In the expression of Shh in the anterior region of fins and conse- pterus fi , inhibiting or activating Shh signaling during n regeneration quently the propterygium acquires the identity of the metaptery- affected the number of radials, in parallel with the effects observed gium (16). This change in identity resembles the transformation of in the number of digits in terrestrial vertebrates, including the radius to ulna of tetrapod limbs treated with RA, Shh, or through posterior–anterior responsiveness to Shh dose. transplantation of the polarizing zone (31, 35, 36). A corollary of the equivalence of radials to metacarpals is that Homology Between Polypterus Pectoral Fin and Tetrapod Limbs: A the autopod is an ancient structure that existed before the Proposal with Evolutive Implications. The detailed comparison of emergence of tetrapods. This contrasts with a commonly accepted Polypterus fi n bone elements with radius/radiale, ulna/ulnare, view in which the autopod is considered a structure appearing de BIOLOGY and intermedium, the mesopodial most conserved elements in novo in tetrapods. The proposed primitive origin of the autopod is DEVELOPMENTAL tetrapod-like fishes and extinct and extant tetrapods, is difficult in agreement with the autopodial-like Hox expression pattern

Cuervo et al. PNAS | March 6, 2012 | vol. 109 | no. 10 | 3841 Downloaded by guest on October 1, 2021 recently determined in Polyodon spathula fin, another basal acti- hybridization protocol used was based on that described by Nieto et al. (42), nopterygian fish (37), and in Scyliorhinus canicula, a member of with two major modifications: proteinase K treatment was extended up to the most basal lineage of jawed vertebrates (38). Also, Hoxd13 45 min for large pieces comprising the cleithrum/scapulocoracoid bones, and expression in the Australian lungfish closely matches the late the last wash in TBST [for 1 L of 10× TBST: 8 g NaCl, 0.2 g KCl, 25 mL of 1 M expression patterns observed in the tetrapod autopod (39). These Tris-HCl (pH 7.5), 10 mL Tween 20] before signal development was per- data together suggest that fin radials and tetrapod digits may be formed for 48 h. Although in general signal development was performed for patterned by shared mechanisms that are distinct from those less than 24 h, occasionally it was necessary to extend it for 48 h; in these patterning the proximal fin/limb bone elements. This gene ex- latter cases, control probes did not show any signal (Fig. S3). Polypterus Shh pression pattern homology has led to the deep homology concept probe was obtained by nested RT-PCR using total RNA from regenerating fi fi in which a specific gene regulation mechanism is used to build ns and the rst-Choice RLM-race Kit (Ambion). After cDNA synthesis, we fi fi ′ structures with no evident phylogenetic or morphologic re- performed the rst ampli cation round using the 3 -RACE outer primer and fi ′ ′ lationship (40). Thus, shared gene expression patterns may not a speci c Shh oligonucleotide 5 -GAGAAGACCYTAGGGGCCAGCGCAGA-3 (based on nucleotide sequence identity among different species including necessarily indicate structural homology or evolutionary origin fi fi fi (41). In the present report we observed that comparative analyses mouse, chick, zebra sh, and paddle sh). In the second round of ampli cation the following oligonucleotides were used: 5′-GGGCCAGCGGCAGATA- of the RA and Shh responses provide additional clues to un- CGAG-3′ and 5′-TAGACCCAGTCAAATCCAGC-3′, which encompass codons derstand appendicular skeleton evolution. Thus, in terms of deep for amino acids 23–136 of mouse Shh. The 348-bp fragment obtained was homology, we must consider, in addition to shared genetic pro- cloned in pTopo2.1 (Invitrogen) and the identity determined by sequencing grams, shared responses of developing/regenerating structures to (GenBank accession no. HM190156). morphogens. The remarkable ability of Polypterus to regenerate fi their pectoral ns will allow a detailed comparative analysis of RA, Cyclopamine, and Shh Agonist Treatment. Bichirs were transferred on small gene expression patterns in combination with growth factor glass containers with 100 mL of water. Cyclopamine (BIOMOL) was adminis- responses and, in this manner, to experimentally test whether the trated by adding it directly to water at 2 μg/mL or 1 μg/mL final concentration Polypterus tribasal structure of is the ancestor of the tetrapod from a 5 mg/mL stock in DMSO. Hh-Ag1.3 (Curis) was used at 100 and 200 nM zeugopod and autopod. from a 1-mM stock in DMSO. Treatments with a commercially available Smoothened agonist SAG (Calbiochem) at 200 nM final concentration gave Materials and Methods similar effects. Equivalent amounts of DMSO were added to controls. Treat- Whole-Mount Skeletal Preparations. Commercially obtained juvenile bichirs, ments were performed for 3 wk starting at 1–9 d (early stages) or 12–18 d ≈ Polypterus senegalus and P. ornatipinnis, 6 cm long shortly after gill re- (late stages) after amputation. Water containing cyclopamine or Hh-Ag1.3 duction phase (2), were used for the experiments. Fishes were maintained in was changed every day during treatment. After treatment, regeneration was glass tanks at 28 °C. Amputations were performed with nail clippers after allowed to continue for a further 3 wk, before fishes were killed and fins anesthesia by immersion in cold water containing a 0.1% tricaine solution. dissected. Amputations were performed closer to the body. Bichirs were killed with an overdose of tricaine; the fins were dissected, fixed and, dehydrated overnight The protocol for RA injection was as reported for urodeles (14). All-trans-RA μ (ON) in 100% ethanol. Fins were incubated in acetone for 24 h and then (Sigma) dissolved in DMSO (15 mg/mL) was injected i.p. (100, 75, or 50 g/g of stained for 3 h at 37 °C plus ON at room temperature in alcian blue and alizarin body weight), on day 3 after amputation. For time-dependant studies, sub- red in 70% ethanol with 5% acetic acid. Fins were rinsed in tap water before sequent injections were done at 6 and 9 d after amputation with 100 μg/g of clearing in 1% KOH and 20% glycerol for 24 h and then in graded glycerol. body weight.

Histology. Regenerates were fixed in modified Karnovsky for 5 h at 4 °C and ACKNOWLEDGMENTS. We thank Dr. Chris Wood for discussion of data and then washed ON in sodium cacodylate buffer at 4 °C. After a postfixation in careful reading of the manuscript; Marcia Bustamante, Concepción Valencia, 1% osmium, samples were dehydrated in graded ethanol series and em- and Sr. José G. Baltazar García for their technical assistance; and Sr. Dionisio bedded in Epon. Half-micrometer sections were stained with toluidine blue 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 and visualized by bright-field microscopy. by Grant IN218607 from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica-Dirección General Asuntos del Personal Académico- Whole-Mount in Situ Hybirdization. Fins were fixed ON with MEMPFA [0.1 M Universidad Nacional Autónoma de México (PAPIIT-DGAPA) and Grant Mops (pH 7.4), 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde]. The in situ 39930-Q from Consejo Nacional de Ciencia y Tecnología (CONACyT).

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