© 2006 Nature Publishing Group
Evidence that mechanisms of fin development
evolved in the midline of early vertebrates
Renata Freitas1, GuangJun Zhang1& Martin J. Cohn1,2
The origin of paired appendages was a major evolutionary
innovation for vertebrates, marking the first step towards fin-
(andlater limb-)driven locomotion.Theearliest vertebratefossils
lack paired fins but have well-developed median fins1,2, suggesting
that the mechanisms of fin development were assembled first in
the midline. Here we show that shark median fin development
involves the same genetic programs that operate in paired appen-
dages. Using molecular markers for different cell types, we show
that median fins arise predominantly from somitic (paraxial)
mesoderm, whereas paired appendages develop from lateral
plate mesoderm. Expression of Hoxd and Tbx18 genes, which
specify paired limb positions3,4, also delineates the positions of
median fins. Proximodistal development of median fins occurs
beneath an apical ectodermal ridge, the structure that controls
outgrowth of paired appendages5–7. Each median fin bud then
acquires an anteroposteriorly-nested pattern of Hoxd expression
similar tothatwhichestablishesskeletalpolarity inlimbs8,9.Thus,
despite their different embryonic origins, paired and median fins
utilize a common suite of developmental mechanisms. We
extended our analysis to lampreys, which diverged from the
lineage leading to gnathostomes before the origin of paired
appendages2,10, and show that their median fins also develop from
results suggest that the molecular mechanisms for fin develop-
ment originated in somitic mesoderm of early vertebrates, and
that the origin of paired appendages was associated with
re-deployment of these mechanisms to lateral plate mesoderm.
Outgrowth of paired fins and limbs is maintained by the apical
ectodermal ridge (AER) at the distal margin of the buds5,6, and
members of the Fgf family synergistically mediate its signalling
activity7,11. In catsharks, median fins develop from a continuous
finfold extending along the dorsal and ventral midlines (Supplemen-
tary Fig. 1). Outgrowth of the median finfold occurs beneath an
tary Fig. 2). The AER then becomes an apical ectodermal fold (AEF),
as in the paired fins of teleosts6. The similar embryology of median
and paired fins raised the possibility that a common set of mecha-
nisms regulates their development, but their anatomical positions
suggested distinctive embryonic origins. Transplantation experi-
ments in amphibians have led to the idea that median finfolds are
neural crest derived, and the zebrafish caudal fin was shown to
originate, at least in part, from trunk neural crest12,13. Recent fate-
mapping studies, however, demonstrated that somitic mesoderm
contributes to amphibian median finfold development14. We there-
fore set out to determine the embryonic origin of catshark median
Studies in several model systems have shown that Foxc2 and Zic1
are expressed in the sclerotome, and that they remain in these cells as
they migrate dorsally around the neural tube to form neural arches
and spinous processes15,16. Neither of these genes is expressed by
migratory trunk neural crest or differentiated myotome15,16, making
them suitable for distinguishing sclerotomal cells during median fin
development. We cloned and examined the expression of catshark
Foxc2 and Zic1 and found that, as in tetrapods, both are expressed
throughout the sclerotome (Fig. 1a, b). During median fin develop-
median finfolds (Fig. 1a, b; Supplementary Figs 3a, b and 4a, b). We
also examined Scleraxis (sclerotome-related helix-loop-helix type
transcription factor), a marker of the sclerotomal sub-compartment
(syndetome) that forms axial tendons in chick and mouse17,18.
Catshark Scleraxis marks a similar subset of the sclerotome and,
like Foxc2 and Zic1, its expression domain extends into the median
all three sclerotomal markers persisted during differentiation of the
fin radials and neural arches.
To determine whether cells from the dermomyotome and neural
crest also participate in median fin development, we examined Pax7,
a marker of these cell types in other vertebrate embryos19. Pax7 was
initially expressed in the catshark dermomyotome and dorsal neural
tube, but Pax7-expressing cells were not detected in the median fin
before stage 31 (Fig. 1d; Supplementary Figs 3d and 4d). Pax7
expression then extended from the dermomyotome into the median
fins, in the muscle projections lateral to the developing skeleton
(Fig. 1d; Supplementary Fig. 3d). Immunolocalization of Zn12, a
neural crest marker20, revealed that a limited number of neural crest
cells also invaded these fins, but most of the mesenchyme was
negative for this marker (Fig. 1e; Supplementary Fig. 3e). By stage
31, Zn12 had localized predominantly to the space within the AEF,
where dermal rays develop, and subjacent to the distal ectoderm
bulk of the median fin mesenchyme is derived from sclerotome,
although cells from dermomyotome and neural crest also contribute
to median fin development (Fig. 1f; Supplementary Fig. 3f). If
technical challenges can be overcome, cell labelling in shark embryos
will further address the contributions of these cell types.
During limb development, lateral plate mesoderm is regionalized
into limb-forming and non-limb-forming domains by differential
expression of Hox and Tbx genes3,4. We investigated whether ante-
roposterior regionalization of the median finfold into dorsal, anal
and caudal regions involves similar mechanisms. In catsharks,
median fins lie posterior to the cloaca, suggesting that, if Hox
genes are involved in their development, then the most likely
candidates would be AbdB-related Hox9–Hox13 genes. Therefore,
we cloned 5
expression during median fin development (Fig. 2 and Supplemen-
tary Figs 5 and 6). Prior to the extension of sclerotome towards the
dorsal and ventral finfolds, we observed collinear expression of
Hoxd9, Hoxd10, Hoxd12 and Hoxd13 in the somitic mesoderm
(Supplementary Fig. 6). The Hoxd9 domain extended anterior to
the cloaca, marking the region in which median fin outgrowth was
0Hoxd genes from catsharks and examined their
1Department of Zoology,2Department of Anatomy and Cell Biology, University of Florida, PO Box 118525, Gainesville, Florida 32611, USA.
Vol 442|31 August 2006|doi:10.1038/nature04984
© 2006 Nature Publishing Group
maintained (Supplementary Figs 1 and 6). For Hoxd9 and Hoxd10,
we observed different anterior boundaries in the neural tube, para-
The mesenchymal component of the finfold had developed by
stage 25, and Hoxd genes were expressed in an anteroposteriorly-
nested pattern along the dorsal and ventral finfolds, with specific
combinations characterizing first dorsal, second dorsal and anal
fin levels (Fig. 2a, b). The first dorsal fin region was characterized
by expression of Hoxd9 and Hoxd10, whereas the second dorsal and
anal regions were distinguished by additional expression of Hoxd12
(Fig. 2a, b). Hoxd13 remained confined to the caudal fin region
(Fig. 2a, b).
As individual fins emerged fromthe finfold, Hoxd gene expression
persisted in the developing fins but was downregulated in the
adjacent somites (Fig. 2c). In the first dorsal fin, Hoxd9 and
Hoxd10 expression was maintained, and Hoxd12 and Hoxd13 were
activated sequentially (Figs 2c and 3a). The second dorsal and anal
finsexpressedHoxd10, Hoxd12 and subsequently Hoxd13, but Hoxd9
These patterns are consistent with the hypothesis that combinatorial
expression of Hoxd genes may establish a molecular map for median
fin position and identity21.
It is unlikely that Hox genes act alone to specify fin and limb
position. Recent work has implicated Tbx18 in defining anterior
boundaries of forelimbs and somites in chick embryos4. To examine
whether this gene may also relate to boundary formation in median
Figure 1 | Developmental origin of catshark median fins. Transverse
sections through first dorsal fins; dorsal is to top. Developmental stage (St.)
indicated at top. a–c, Expression of Foxc2 (a), Zic1 (b) and Scleraxis (c) in
sclerotome surrounding the neural tube (Nt), and in dorsal midline
mesenchyme invading the median fins. At stage 31, the strongest expression
of Foxc2 and Scleraxis is detected in the developing fin radials (R). d, Pax7
expression in the dorsal lip of the dermomyotome (arrows, stages 29 and
30), and later in myotomal projections invading the median fins (arrows,
fin mesenchyme and within the apical ectodermal fold (AEF). f, Schematic
summary of the cellular contributions to the dorsal median fins.
Figure 2 | Regionalized expression of Hoxd genes and Tbx18 along the
median finfold of catsharks. Developmental stage indicated in lower left
corners. Dorsal is to top in sections and left in whole-mounts. a, Hoxd gene
dorsal (1D), second dorsal (2D) and anal (A) fins. Red dotted lines indicate
anterior expression boundaries in the median finfold, as verified by
histological sections. b, Transverse sections immediately posterior to dotted
lines in a showing Hoxd gene expression in dorsal and ventral midline
mesenchyme beneath the AEF (arrowheads). Pel, pelvic fin; Cl, cloaca;
Nt, neural tube. c, Hoxd gene expression during emergence of individual
median fins at stage 30. Arrowheads mark anterior and arrows mark
posterior boundaries of Hoxd expression in dorsal and ventral finfolds.
d, e, Expression of Tbx18 in somites (S) in whole-mount (d) and transverse
section (e). f, Tbx18 expression becomes detectable in presumptive dorsal
and anal fins at stage 26 (arrows). g, Transverse section showing Tbx18
expression in first dorsal fin (arrows) and in sclerotome (arrowheads).
NATURE|Vol 442|31 August 2006
© 2006 Nature Publishing Group
fins, we cloned and examined expression of a catshark Tbx18
orthologue. Tbx18 was first expressed in the anterior region of
each somite (Fig. 2d, e). During finfold outgrowth, we detected
Tbx18 in three discrete domains that delineated the prospective first
dorsal, second dorsal and the anal fins (Fig. 2f, g), resembling the
pattern observed in chick limbs4. Given the function of Tbx18 in
specifying limb position in lateral plate mesoderm, we suggest that
Tbx18 may also participate in specification of median fin position
paired fin development.
Dorsal and anal fin skeletons are polarized along their antero-
posterior axes (Supplementary Fig. 1d, e). In paired limb buds, Hoxd
genes are expressed along the anteroposterior axis in a spatially and
temporally collinear pattern that determines the polarity of the
skeleton8,9. We therefore explored whether Hoxd expression in each
median fin follows the patterns observed in paired limbs. In the first
dorsal fin, Hoxd9 and Hoxd10 were expressed broadly from the onset
of budding (Fig. 3a). Hoxd12 became detectable posteriorly by stage
31, and Hoxd13 expression was observed nested within the Hoxd12
domain one stage later (Fig. 3a). Similar collinear patterns were
observed in the second dorsal and anal fins (Fig. 3b, c). Thus, shark
dorsal and anal fins exhibit the characteristic collinearity of paired
appendages22,23. In contrast to this ‘appendicular’ pattern of
expression in dorsal and anal fins, the caudal fin develops in the
absence of Hoxd gene expression after stage 30 (Fig. 3d). Posterior
polarizing activity in paired limbs, which patterns the anteroposter-
ior axis via secretion of Sonic hedgehog8. Our finding of a conserved
relationship between the polarity of Hoxd gene expression and the
anteroposterior pattern of the fin skeleton suggests that a similar
mechanism may operate in median fins.
Interpretation of these results in the context of fin evolution
suggests that the fin development program may have originated in
paraxial mesodermally-derived median fins before paired fins
evolved in lateral plate mesoderm. To test this hypothesis, we
Figure 3 | Anteroposterior nesting of Hoxd gene expression in catshark
median fin buds. Anterior is to top and dorsal is to left in whole-mounts;
dorsal is to top in sections. Stages of development indicated in lower left
corners. Diagram to left shows location of fins (green) depicted in adjacent
panels. a, Hoxd expression in first dorsal fins. Arrows point to Hoxd12 and
Hoxd13 expression at the posterior margin of fin. b, Hoxd expression in
second dorsal fins. Asterisk marks the posterior boundary of Hoxd9
expression that has started to shift anteriorly. Arrows point to expression of
Hoxd expression in anal fins, and lower panels show transverse sections
through these fins. Arrowheads indicate Hoxd9 and Hoxd10 expression
along the proximal region of fin. Arrows mark posterior expression of
Hoxd12 and Hoxd13. Note temporal and spatial collinearity of Hoxd gene
expression within the dorsal (a, b) and anal (c) fins. d, Caudal fins lacking
Hoxd expression at stage 30; arrows indicate posterior limits of Hoxd
expression in the pre-caudal finfolds.
Figure 4 | Lamprey median fin development. Stages of development
indicated in lower left corners. a–e, j and k are transverse sections with
dorsal to top, f–i are whole-mounts with dorsal to left. a, Expression of
finfold. b, Parascleraxis expression has expanded into the dorsal finfold
(arrowheads). c, Zn12 staining in two clusters of cells at the base of the
dorsal finfold (arrowheads). d, e, Expression of Hox9y (d) and Hox10w (e)
in the dorsal finfold mesenchyme (arrowheads). Note that the fin tissue
distal to the expression domains is ectodermal. f, g, Expression of Hox9y (f)
and Hox10w (g) in the finfold (arrowheads). Dashed lines mark the
approximate planes of sections shown in d and e. Cl, cloaca. h, Expression
of Tbx15/18 in the anterior part of each somite (S; arrows) and in the
dorsal finfold (arrowheads) at stage 24. i, Expression of Tbx15/18 in the
median finfold at stage 26 (arrowheads). Dashed lines mark approximate
planes of sections shown in j and k. j, k, Transverse sections taken anterior
(j) and posterior (k) to the expression boundary of Tbx15/18 in the
median finfold. Note that somitic expression extends anterior to the finfold
NATURE|Vol 442|31 August 2006
© 2006 Nature Publishing Group
extended our analysis to lampreys, which exhibit the plesiomorphic
condition of median fins in the absence of paired appendages2,10.
During lamprey embryogenesis, a single ectodermal median finfold
develops along the entire trunk. Proximodistal expansion of finfold
mesenchyme occurs predominantly in the posterior region, where
median fins differentiate during metamorphosis24. To determine
whether median fins of lampreys and sharks have the same embryo-
nic origin, we isolated and characterized the expression of a lamprey
Scleraxis orthologue. Phylogenetic analysis of our full-length clone
placed it as the sister to the gnathostome Scleraxis/Paraxis clade, and
therefore we designated it Parascleraxis (Supplementary Fig. 7).
Parascleraxis expression was detected at stage 26 in sclerotomal
cells adjacent to the neural tube but not in dermomyotome
(Fig. 4a). By stage 28, the Parascleraxis domain extended into the
median finfold, consistent with a sclerotomal contribution to lam-
prey medianfins(Fig.4b).Restriction ofParascleraxis tothelamprey
sclerotome also suggests that the dermomyotomal domain of Paraxis
in gnathostomes may be a novel site of expression that was acquired
after duplication of the ancestral Parascleraxis gene gave rise to
Paraxis and Scleraxis. We then stained lamprey embryos for Zn12,
as previous workers reported migration of neural crest cells into the
lamprey median finfold25,26. Zn12 signal was detected in two clusters
of sub-epidermal cells at the base of the finfold, and later in a narrow
column of cells at its distal tip, but the bulk of median finfold
mesenchyme was negative for Zn12 (Fig. 4c and data not shown).
These data suggest that, as in sharks, the sclerotome and a limited
number of neural crest cells give rise to the median fin mesenchyme
We next asked whether anteroposterior boundaries of median fins
in lampreys and sharks are specified by an evolutionarily conserved
mechanism involving 5
the expression of lamprey Hox9y and Hox10w (ref. 27), and detected
The anterior boundary of Hox9y expression in the dorsal finfold and
adjacent somites extended anterior to the Hox10w domain and
larval development (Fig. 4f, g). We also screened for a lamprey Tbx18
orthologue and isolated a complementary DNA fragment that our
phylogenetic analyses joined to the base of the gnathostome Tbx15/18
clade (Supplementary Fig. 8). Lamprey Tbx15/18 was expressed ante-
riorly in each somite and in the median finfold at stage 24 (Fig. 4h).
the fin-forming region, whereas the somitic expression extended
along the entire trunk at stage 26 (Fig. 4i–k).
Conservation of the embryonicorigin and the patterns of Hox and
Tbx gene expression in shark and lamprey median fins suggests that
before the origin of paired fins. Our finding that median fin
mesenchyme arises predominantly from somites suggests that these
cells may acquire their positional identities, in the form of Hox and
Tbx expression, during regionalization of paraxial mesoderm. We
suggest that the origin of paired appendages from lateral plate
mesoderm involved re-deployment of mechanisms that were
originally restricted to paraxial mesoderm, where they regulated
development of cartilage (Hox9–Hox13, Tbx18), muscle (Pax7,
Paraxis) and tendon (Scleraxis) in the axial skeleton and median
fins. Reports of Msx and Dlx expression in paired and median fins of
zebrafish28,29may reflect additional evolutionary signatures of this
co-option. It is possible that the mechanisms of fin and limb
development were established in median finfolds even before the
origin of vertebrates. Analysis of median finfold development
in cephalochordates will further test the hypothesis that these
mechanisms emerged early in chordate evolution.
0Hox and Tbx18 orthologues. We analysed
with reverse transcription (RT–PCR) was performed to amplify catshark
fragments of 5
Hoxd12, 561bp; Hoxd13, 579bp), Tbx18 (534bp), Zic1 (204bp), Foxc2
(267bp) and Pax7 (294bp) using cDNA from a stage 28 Scyliorhinus canicula.
A full-lengthcopyof catshark Scleraxis(1,413bp) wasobtainedbyRT–PCRand
Parascleraxis) were isolated by RT–PCR from a Petromyzon marinus cDNA
library (a gift from J. Langeland) using Advantage GC-PCR Kit (Clontech). The
Cloning Vector (Qiagen). Orthology of the cloned sequences was determined
by protein alignment comparisons followed by maximum-likelihood and
Whole-mount in situ hybridization and immunochemistry. These were
performed as described previously30. Antibodies against Fgf8 (Santa Cruz
Biotechnology Inc), Distal-less (Dll/Dlx; kindly supplied by G. Boekhoff-Falk)
and Zn12 (Developmental Studies Hybridoma Bank) were diluted to working
concentrations of 1:100, 1:70 and 1:5 respectively. Peroxidase-conjugated
secondary antibodies (DAKO) were diluted to 1:500 in phosphate buffered
saline (PBS) with 1% Triton and 1% serum. Following whole-mount in situ
hybridization or immunochemistry, the specimens were equilibrated in graded
sucrose in PBS (15% and 30%) at 48C and graded gelatine in PBS (20% gelatine
in 30% sucrose and 20% gelatine) at 508C. Embryos were then mounted in
Tissue-Tek OCT (Sakura Finetek) and cryosectioned at 20–35mm.
0Hoxd genes (Hoxd9, 387base pairs, bp; Hoxd10, 813bp;
0rapid amplification of cloned ends (RACE). Lamprey genes (Tbx15/18 and
Received 26 July 2005; accepted 19 June 2006.
Published online 26 July 2006.
1. Zhang, X. G. & Hou, X. G. Evidence for a single median fin-fold and tail in the
Lower Cambrian vertebrate, Haikouichthys ercaicunensis. J. Evol. Biol. 17,
1162– -1166 (2004).
Coates, M. I. The origin of vertebrate limbs. Development (Suppl.), 169– -182
Cohn, M. J. et al. Hox9 genes and vertebrate limb specification. Nature 387,
97– -101 (1997).
Tanaka, M. & Tickle, C. Tbx18 and boundary formation in chick somite and
wing development. Dev. Biol. 268, 470– -480 (2004).
Saunders, J. W. The proximo-distal sequence of origin of parts of the chick
wing and the role of the ectoderm. J. Exp. Zool. 108, 363– -403 (1948).
Grandel, H. & Schulte-Merker, S. The development of the paired fins in the
zebrafish (Danio rerio). Mech. Dev. 79, 99– -120 (1998).
Grandel, H., Draper, B. W. & Schulte-Merker, S. dackel acts in the ectoderm of
the zebrafish pectoral fin bud to maintain AER signaling. Development 127,
4169– -4178 (2000).
Zakany, J., Kmita, M. & Duboule, D. A dual role for Hox genes in limb anterior-
posterior asymmetry. Science 304, 1669– -1672 (2004).
Tarchini, B. & Duboule, D. Control of Hoxd genes collinearity during early limb
development. Dev. Cell 10, 93– -103 (2006).
10. Donoghue, P. C. J., Forey, P. L. & Aldridge, R. J. Conodont affinity and chordate
phylogeny. Biol. Rev. Camb. Philos. Soc. 75, 191– -251 (2000).
11.Sun, X., Mariani, F. V. & Martin, G. R. Functions of FGF signalling from the
apical ectodermal ridge in limb development. Nature 418, 501– -508 (2002).
12. Tucker, A. S. & Slack, J. M. Independent induction and formation of the dorsal
and ventral fins in Xenopus laevis. Dev. Dyn. 230, 461– -467 (2004).
13. Smith, M., Hickman, A., Amanze, D., Lumsden, A. & Thorogood, P. Trunk
neural crest origin of caudal fin mesenchyme in the zebrafish Brachydanio rerio.
Proc. R. Soc. Lond. B 256, 137– -145 (1994).
14. Sobkow, L., Epperlein, H. H., Herklotz, S., Straube, W. L. & Tanaka, E. M. A
germline GFP transgenic axolotl and its use to track cell fate: dual origin of the
fin mesenchyme during development and the fate of blood cells during
regeneration. Dev. Biol. 290, 386– -397 (2006).
15. Furumoto, T. A. et al. Notochord-dependent expression of MFH1 and PAX1
cooperates to maintain the proliferation of sclerotome cells during the
vertebral column development. Dev. Biol. 210, 15– -29 (1999).
16. Sun Rhodes, L. S. & Merzdorf, C. S. The zic1 gene is expressed in chick
somites but not in migratory neural crest. Gene Expr. Patterns 6, 539– -545
17. Brent, A. E., Schweitzer, R. & Tabin, C. J. A somitic compartment of tendon
progenitors. Cell 113, 235– -248 (2003).
18. Brent, A. E. & Tabin, C. J. FGF acts directly on the somitic tendon progenitors
through the Ets transcription factors Pea3 and Erm to regulate scleraxis
expression. Development 131, 3885– -3896 (2004).
19. Lacosta, A. M., Muniesa, P., Ruberte, J., Sarasa, M. & Dominguez, L. Novel
expression patterns of Pax3/Pax7 in early trunk neural crest and its
melanocyte and non-melanocyte lineages in amniote embryos. Pigment Cell
Res. 18, 243– -251 (2005).
20. Trevarrow, B., Marks, D. L. & Kimmel, C. B. Organization of hindbrain segments
in the zebrafish embryo. Neuron 4, 669– -679 (1990).
21. Mabee, P. M., Crotwell, P. L., Bird, N. C. & Burke, A. C. Evolution of median fin
modules in the axial skeleton of fishes. J. Exp. Zool. 294, 77– -90 (2002).
22. Sordino, P., Van der Hoeven, F. & Duboule, D. Hox gene expression in teleost
fins and the origin of vertebrate digits. Nature 375, 678– -681 (1995).
NATURE|Vol 442|31 August 2006
© 2006 Nature Publishing Group Download full-text
23. Nelson, C. E. et al. Analysis of Hox gene expression in the chick limb bud.
Development 122, 1449– -1466 (1996).
24. Richardson, M. K. & Wright, G. M. Developmental transformations in a normal
series of embryos of the sea lamprey Petromyzon marinus (Linnaeus). J. Morphol.
257, 348– -363 (2003).
25. Hirata, M., Ito, K. & Tsuneki, K. Migration and colonization patterns of HNK-1-
immunoreactive neural crest cells in lamprey and swordtail embryos. Zool. Sci
14, 305– -312 (1997).
26. McCauley, D. W. & Bronner-Fraser, M. Neural crest contributions to the
lamprey head. Development 130, 2317– -2327 (2003).
27. Force, A., Amores, A. & Postlethwait, J. H. Hox cluster organization in the
jawless vertebrate Petromyzon marinus. J. Exp. Zool. 294, 30– -46 (2002).
28. Akimenko, M. A., Ekker, M., Wegner, J., Lin, W. & Westerfield, M.
Combinatorial expression of three zebrafish genes related to distal-less: part of
a homeobox gene code for the head. J. Neurosci. 14, 3475– -3486 (1994).
29. Akimenko, M. A., Johnson, S. L., Westerfield, M. & Ekker, M. Differential
induction of four msx homeobox genes during fin development and
regeneration in zebrafish. Development 121, 347– -357 (1995).
30. Freitas, R. & Cohn, M. J. Analysis of EphA4 in the lesser spotted catshark
identifies a primitive gnathostome expression pattern and reveals co-option
during evolution of shark-specific morphology. Dev. Genes Evol. 214, 466– -472
Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank A. Burke, P. Mabee, P. Crotwell and B. Shockey
for commenting on the manuscript, A. Graham for sharing reagents, and L. Page
and G. Weddle for assistance with lamprey collection. R. Freitas is a PhD
student of the GABBA Program (ICBAS, Univ. Oporto) and was supported by a
fellowship from FCT, Praxis XXI.
Author contributions R.F. performed and designed (with M.J.C.) the reported
studies. G.Z. performed part of the gene cloning and phylogenetic analyses.
M.J.C. supervised the research project, and assisted in the experimental design.
R.F. and M.J.C. wrote the manuscript. All authors discussed the results and
commented on the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Sequences for Foxc2, Zic1, Scleraxis, Pax7, Hoxd9,
Hoxd10, Hoxd12, Hoxd13 and Tbx18 from S. canicula, and Parascleraxis and Tbx15/
18 from P. marinus, are deposited in GenBank under accession numbers
DQ659101–DQ659111. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to M.J.C.
NATURE|Vol 442|31 August 2006