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Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved November 2, 2009 (received for review September 16, 2009)
Abstract
Paired fins/limbs are one of the most successful vertebrate innovations, since they are used for numerous fundamental activities, including locomotion, feeding, and breeding. Gene duplication events generate new genes with the potential to acquire novel functions, and two rounds of genome duplication took place during vertebrate evolution. The cephalochordate amphioxus diverged from other chordates before these events and is widely used to deduce the functions of ancestral genes, present in single copy in amphioxus, compared to the functions of their duplicated vertebrate orthologues. The T-box genes Tbx5 and Tbx4 encode two closely related transcription factors that are the earliest factors required to initiate forelimb and hind limb outgrowth, respectively. Since the genetic components proposed to be responsible for acquiring a trait during evolution are likely to be involved in the formation of that same trait in living organisms, we investigated whether the duplication of an ancestral, single Tbx4/5 gene to give rise to distinct Tbx4 and Tbx5 genes has been instrumental in the acquisition of limbs during vertebrate evolution. We analyzed whether the amphioxus Tbx4/5 gene is able to initiate limb outgrowth, and assayed the amphioxus locus for the presence of limb-forming regulatory regions. We show that AmphiTbx4/5 is able to initiate limb outgrowth and, in contrast, that the genomic locus lacks the regulatory modules required for expression that would result in limb formation. We propose that changes at the level of Tbx5 and Tbx4 expression, rather than the generation of novel protein function, have been necessary for the acquisition of paired appendages during vertebrate evolution.
Since Ohno's visionary hypothesis concerning the origin of vertebrate innovations by genome duplication (1), numerous reports have been published supporting this concept (reviewed in ref. 2). The cephalochordate amphioxus, a limbless extant invertebrate relative of the vertebrates (3), has been extensively used to study the ancestral functions of genes, present in single copy in amphioxus, that have been duplicated in the vertebrate lineage. Amphioxus exhibits many basal chordate characteristics, including the presence of a dorsal nerve cord, a notochord, and segmented paraxial mesoderm, but lacks many vertebrate characteristics such as migratory neural crest cells, a cranium, or an endoskeleton (4). We sought to investigate the origin of one of the most successful vertebrate innovations, paired appendages, which include the pectoral and pelvic fins of fish and their derived homologues, the forelimbs and hind limbs of tetrapods.
The T-box genes Tbx4 and Tbx5 are paralogous genes that arose by duplication of a single, ancestral Tbx4/5 gene. Extant amphioxus possesses a single Tbx4/5 gene (AmphiTbx4/5) (5) and lacks paired appendages, whereas all jawed vertebrates with two pairs of paired appendages have distinct, postduplication Tbx4 and Tbx5 genes. In vertebrates, Tbx5 is expressed in the lateral plate mesoderm (LPM) of the presumptive pectoral fin/forelimb region, whereas Tbx4 is expressed in the pelvic fin/hind limb region (reviewed in ref. 6). Tbx5 and Tbx4 are the earliest factors required for the initiation of limb outgrowth (7–10), and are sufficient to initiate outgrowth in an otherwise limbless state (11, 12). Both genes encode transcription factors that directly regulate the expression of Fibroblast growth factor-10 (Fgf10) and establish an FGF signaling loop that drives limb outgrowth (7, 9).
To investigate the relationship betwen duplication of a single, ancestral, Tbx4/5 locus to give rise to separate Tbx5 and Tbx4 loci, and the acquisition of vertebrate paired appendages, we compared the functions of the amphioxus Tbx4/5 gene, and its genomic regulatory landscape, to those of the mouse Tbx4 and Tbx5 gene loci. A priori, one can envisage two alternative scenarios to explain the origin of paired appendages following duplication of the single Tbx4/5 gene. First, before duplication of the Tbx4/5 gene, mutations within the coding region endowed the descendant paralogous genes, Tbx5 and Tbx4, with the potential to initiate limb outgrowth; specifically, the proteins acquired the ability to activate Fgf10 in the LPM. Second, that after the divergence of cephalochordates, mutation(s) in the regulatory regions of the ancestral Tbx4/5 locus, led ultimately to the expression of the vertebrate Tbx4 and Tbx5 genes in the early LPM before limb bud stages, when this tissue is competent to initiate limb outgrowth (13, 14). To investigate this further, we undertook two complementary approaches: first, we tested the potential of the amphioxus Tbx4/5 protein to initiate limb outgrowth in the forelimbless Tbx5 conditional knockout mouse (10) and second, we tested the ability of the AmphiTbx4/5 and mouse Tbx5 and Tbx4 genomic regions to drive gene expression in the early LPM by transient transgenesis experiments in the mouse.
Results
We have used our limb-rescue assay (11) to test the ability of the amphioxus Tbx4/5 protein to initiate limb outgrowth. We generated three independent transgenic lines (Prx1-Amphi4/5#1–3) in which the expression of the full-length AmphiTbx4/5 cDNA was driven under the control of the Prx1 promoter (Fig. 1 A and B) (15) in early, presumptive forelimb, LPM. In these transgenic lines the amphioxus gene is ectopically expressed in forelimbs as well as in hind limbs (Fig. 2 A–C). Analysis of e17.5-rescued embryos (Tbx5lox/lox; Prx1-Cre; Prx1-Amphi4/5) revealed that AmphiTbx4/5 is able to initiate and maintain limb outgrowth (Fig. 2 A″–C″) as judged by comparison to the “no-forelimb” phenotype of the Tbx5 conditional knock-out (Fig. 3A′). Skeletal preparations of these embryos showed that all of the skeletal elements along the entire proximo-distal axis of the limb (scapula, stylopod, zeugopod, and autopod) were present in the AmphiTbx4/5-rescued limbs (Fig. 2 A″–C″). The extent of rescue, from partial (Fig. 2B) to complete (Fig. 2 A and C) is presumably dependant on the amount of AmphiTbx4/5 protein expressed in each independent line. No effect of ectopic amphiTbx4/5 was observed in the hind limb (Fig. 2 A″–C″). As additional controls, we generated transgenic lines in which the mouse Tbx4, Tbx5, or chimeric, domain-swap forms of Tbx4 and Tbx5, were driven by the same Prx1 promoter. All of the rescued embryos for these genotypes had forelimbs (Fig. 3) consistent with Tbx4 and Tbx5 having common roles in limb initiation, but not having roles in limb-type specification (11). From these experiments, we conclude that the single AmphiTbx4/5 gene product has the ability to initiate limb outgrowth when expressed at the correct time and in the appropriate tissue (i.e., the prelimb bud stage LPM). These data demonstrate that mutations at the level of the coding sequence cannot account for the limb-forming potential of the mouse and other vertebrate Tbx5 and Tbx4 proteins.
Fig. 1.
Cloning of the amphioxus Tbx4/5 cDNA. (A) Alignment of the deducted amino acid sequence of the AmphiTbx4/5 protein to the mouse Tbx5 and Tbx4 proteins. The N-terminal domain is underlined in blue, the T-domain is underlined in red, and the C-terminal domain is underlined in green. (B) Schematic representation of the Prx1-driven transgenic lines (15). (C) Schematic representation of the Tbx5 and Tbx4 genomic regions cloned in the BGZA reporter vector (16). e1, exon1; e2, exon2; i1, intron1; i2, intron2.
Fig. 2.
The single amphioxus Tbx4/5 gene rescues forelimb outgrowth. (A–C) Whole-mount in situ hybridization to e10.5 Prx1-Amphi4/5 transgenic embryos for the amphioxus Tbx4/5 cDNA (all are dorsal views with anterior to the top). (A′–C′) Rescued e17.5 embryos (all are lateral views of the right side). (A″–C″) skeletal preparations (33) showing the presence of skeletal elements in the Tbx4/5-rescued right forelimbs. au, autopod; fl, forelimb; hl, hind limb; sc, scapula; st, stylopod; z, zeugopod.
Fig. 3.
Mouse Tbx4, Tbx5, or chimeric domain-swap forms of Tbx4 and Tbx5 rescue forelimb outgrowth. (A) Whole-mount in situ hybridization for Fgf10 to e9.5 Tbx5 conditional knock-out embryo showing expression in hind limbs and lack of expression in the forelimb region (arrow). (B–G) Whole-mount in situ hybridization to e10.5 Prx1-transgenic embryos. Probes used are the C-terminal domain of mTbx5 for B, E, and F, and the C-terminal domain of mTbx4 for C, D, and G. (A′–G′) e17.5 embryos showing rescue of forelimb outgrowth in all cases (B′–G′) in comparison to the absence of a forelimb in the Tbx5 conditional knock-out (A′). (A″–G″) Skeletal preparations showing the presence of all forelimb elements in all of the rescued embryos (B″–G″) in comparison to the lack of bones in the Tbx5 conditional knock-out (A″). A schematic representation of the transgenic construct used for the rescue, as well as the genotypes of the rescued embryos are indicated. The orientations shown are the same as in Fig. 2. au, autopod; fl, forelimb; hl, hind limb; sc, scapula; st, stylopod; z, zeugopod.
An alternative scenario is that changes at the level of cis-regulatory regions could account for the acquisition of limbs during vertebrate evolution. To address this, we compared the potential of the amphioxus Tbx4/5 genomic region to drive gene expression in the LPM, at stages when this territory is competent to form a limb, to equivalent genomic regions of the murine Tbx5 and Tbx4 loci. As positive controls, we first sought to locate the mouse regulatory regions required to drive expression of Tbx5 in forelimb LPM and Tbx4 expression in hind limb LPM. To this end, we cloned genomic DNA fragments that span regions upstream of exon3 of the murine Tbx5 and Tbx4 genes into the LacZ reporter vector BGZA (Fig. 1C) (16) for transient transgenesis assays in the mouse. Regulatory elements present <12 Kilobase pairs (Kb) upstream of exon3 of the mouse Tbx5 gene are sufficient to drive expression of the LacZ reporter in the e9.5 forelimb LPM (Fig. 4A). Similarly, regulatory sequences <11 Kb upstream of exon3 of mouse Tbx4 are sufficient to drive expression of the reporter in the e9.5 hind limb LPM (Fig. 4B). When an equivalent region from the AmphiTbx4/5 locus (Bf4/5–10) was used for transgenesis, expression of the reporter in the mouse LPM was never observed (0/12 positive transgenic embryos). To determine whether any LPM-regulatory region was located further away from the amphioxus Tbx4/5 gene, we assayed a bacterial artificial chromosome (BAC; CH302 78M15) containing the entire AmphiTbx4/5 coding region, and 73.3 Kb and 72.3 Kb of flanking region upstream and downstream, respectively, in transient transgenic mice. Whole-mount in situ hybridization on 15 e9.5 transgenic embryos using the full-length amphioxus Tbx4/5 as a probe, showed that this BAC did not contain the regulatory regions capable of driving expression in the prelimb bud LPM. These experiments indicate that the amphioxus Tbx4/5 genomic region lacks the regulatory elements required to drive gene expression in early, vertebrate LPM, the domain in which Tbx5 and Tbx4 are required to execute their limb-forming function.
Fig. 4.
Lateral plate mesoderm expression driven by the murine Tbx5 and Tbx4 promoter region and expression of AmphiTbx4/5. (A and B) b-galactosidase activity staining of an m5–10 (A) and m4–10 (B) e9.5 transgenic embryo. (C) Fifty-six-hour amphioxus larva showing expression of AmphiTbx4/5. Mouse embryos are shown in lateral views of the right side and the amphioxus larva of the left side. (D) Proposed evolutionary scenario leading to the acquisition of paired appendages during vertebrate evolution. The ancestral, single Tbx4/5 gene is represented by a purple box and the postduplicative Tbx5 and Tbx4 genes are indicated by a red and a blue box, respectively. The orange circle represents the ancestral heart enhancer, whereas the green circle shows the acquired LPM enhancer. A schematic representation of the chordate ancestor and a modern fish-like creature are drawn showing expression in the heart territory (orange) and in the fin territory (green). fl, forelimb LPM; H, heart; hl, hind limb LPM; LPM, lateral plate mesoderm.
Finally, we analyzed the expression of AmphiTbx4/5 during amphioxus development (Fig. 4C and ref. 17). Expression of this gene was not observed in embryonic stages. Expression was first detected in late larval (56-h) stages in a posterior, ventral mesoderm domain (arrow in Fig. 4C).
Discussion
It is widely accepted that two rounds of whole genome duplication took place early during vertebrate evolution (18). This genetic material can endow the organism with novel traits, such as in the case of vertebrates, the so-called vertebrate innovations, including two sets of paired appendages. The cephalochordate amphioxus diverged from other chordates before these genome duplications took place and may therefore be used to deduce the functions of ancestral genes, present in single copy in amphioxus, as compared to the functions of their duplicated vertebrate orthologues. The T-box genes Tbx5 and Tbx4 are required to initiate forelimb and hind limb outgrowth, respectively. In the absence of Tbx5, forelimbs do not form, and hind limbs fail to develop in the absence of Tbx4 (9, 10). Amphioxus lacks paired appendages and possesses a single Tbx4/5 gene (AmphiTbx4/5), whereas all vertebrates with paired appendages (from sharks to tetrapods) express Tbx5 in the rostral pair and Tbx4 in the caudal pair (19, 20). We show here that the single AmphiTbx4/5 is able to initiate limb outgrowth when expressed in the correct temporal and spatial domains in mice. We propose that changes at the level of the regulation of Tbx5 and Tbx4 expression, rather than the generation of novel protein function, was necessary for the acquisition of paired appendages during vertebrate evolution.
We also show that in extant amphioxus AmphiTbx4/5 is expressed in a posterior, ventral mesoderm domain (Fig. 4C) of the 56-h larva. Expression in this domain is consistent with the two following evolutionary scenarios that implicate changes at the level of regulatory elements. The ventral mesoderm is where the rudimentary amphioxus heart tube forms, and the amphioxus orthologues of many other genes implicated in heart development in vertebrates, including Nk2-tinman and BMP2/4 (21, 22), are expressed in this domain. Therefore, the first scenario is that the Tbx4/5 gene may have played an ancestral role in heart development and this function has been retained by the vertebrate Tbx5 and Tbx4 genes, since both are expressed in the heart anlagen and are required for heart development (23–26). This implies that, during evolution of the Tbx4/5 subfamily, mutations occurred at the level of their cis-regulatory regions that account for the acquisition of a expression domain in LPM, where Tbx5 and Tbx4 can execute their limb-forming function. A schematic representation of this evolutionary scenario at the Tbx4/5 locus is presented in Fig. 4D. Alternatively, a few reports have suggested that cephalochordate ventral mesoderm could be homologous to vertebrate LPM (2, 27). Hence, a second scenario is that the ancestral Tbx4/5 gene was already expressed in ventral LPM-like tissue but at a later time point during development than its derived vertebrate counterparts, when it is not competent to initiate outgrowth. This suggests that before the duplication of this gene in the vertebrate lineage, but after the divergence of cephalochordates, acquisition of an early enhancer led to the activation of these genes at stages, and in regions, of the LPM that are competent to form a limb.
Functional studies on amphioxus embryos have been severely hampered by the short length of the breeding season and, consequently, the limited timeframe during which experimental manipulations can be performed. Recently, however, progress has been made in the induction of spawning in captive animals, which should greatly facilitate the use of amphioxus as an experimental system (28). It will be fascinating to determine whether the ventral mesoderm of amphioxus larvae has any outgrowth potential, and, if so, to investigate the temporal window of this competence, similar to previous experiments, which show that an exogenous source of FGF applied to the chick flank can induce ectopic limb formation (13, 14).
Materials and Methods
Embryos and Transgenic Lines.
Mouse embryos were staged according to Kaufman (29). Noon on the day a vaginal plug was observed was taken to be e0.5 days of development. Transgenic lines were generated by the Procedural Services Section, NIMR. C57BL/10XCBA/Ca F1 hybrid founders were crossed onto an MF1 background. For pronuclear injection, plasmid DNA was injected at a concentration of 2 ng/mL, while BAC DNA was at 1 ng/μL. To generate rescued embryos (Tbx5lox/lox; Prx1-Cre; Prx1-gene-of-interest) Tbx5lox/+; Prx1-Cre; Prx1-gene-of-interest mice were crossed to homozygote Tbx5lox/lox mice. The conditional Tbx5 allele, the Prx1-Cre transgene and the whole-mount in situ protocol have been described elsewhere (11). Amphioxus embryos were collected as described in ref. 30.
Cloning of Full-Length cDNAs and Chimeras.
The full-length sequence of the AmphiTbx4/5 gene was deduced from the sequence of the CH302 78M15 BAC (17). We used RT-PCR on a 56-h embryonic cDNA sample to obtain the putative full-length coding region. This PCR product was cloned into pSlax to tag it with the flag epitope. For the murine Tbx4 and Tbx5, as well as the chimeric constructs, we used plasmids containing Tbx4 and Tbx5 cDNAs as templates (a gift from Benoit Bruneau, Gladstone Institute, San Francisco, CA). All these PCR products were also cloned in pSlax to add the flag epitope 3′ to the ORFs. We used Advantage HF2 polymerase (Clontech) to perform all of the PCR reactions, and all constructs were sequenced. Primer sequences and PCR conditions are available on request. To produce transgenic constructs, the flag-tagged cDNAs were isolated from pSlax and cloned into the Prx1 promoter construct as described in ref. 11. The schematic representation of the murine constructs in Fig. 3 is as follows: full-length Tbx5 (B) is represented with a blue box, and full-length Tbx4 (C) is shown as a green box. The abbreviations for the chimeric constructs are as follows: 5N4T4C (D) is composed of the N-terminal domain of Tbx5 and the T- and C-terminal domains of Tbx4. 4N4T5C (E) contains the N- and T-domains of Tbx4 and the C-terminal domain of Tbx5. 4N5T5C (F) contains the N-terminal domain of Tbx4 and the T- and C-terminal domains of Tbx5. 5N5T4C (G) contains the N- and T-domain of Tbx5 and the C-terminal domain of Tbx4. See Fig. 1 for the subdivision of murine Tbx5 and Tbx4 in these domains.
Cloning of the Regulatory Regions.
Genomic regions m5–10, m4–10, and Bf4/5–10 were cloned using long-range PCR on BACs containing the mouse Tbx5 (RP-150D8), mouse Tbx4 (RP23–48A17), or amphioxus Tbx4/5 (CH302–78M15) genes, respectively. These regions were subcloned into the BGZA reporter vector (16) using standard techniques. All constructs were verified by sequencing.
Whole-Mount in Situ Hybridization.
Whole-mount in situ hybridization of mouse embryos was performed as described in ref. 31 and ref. 32 for amphioxus embryos. Hybridized mouse embryos were photographed using a Leica digital camera attached to a Leica dissecting scope. Amphioxus embryos were photographed with a Q-Imaging camera attached to a Zeiss Axiophot microscope under DIC/Nomarski optics.
Acknowledgments
We thank David E. K. Ferrier and Peter Osborne for help and material for the amphioxus in situs. We are indebted to Sophie Wood, Procedural Services, National Institute for Medical Research (NIMR) for pronuclear injections, staff from Biological Services, NIMR for mouse husbandry, and Jo Del Buono, Ania Kucharska, Amy Horton, and Ed Oates for technical help and exchange of constructs. This work was supported by the European Molecular Biology Organization and Medical Research Council (C.M.).
Footnotes
- 3To whom correspondence should be addressed. E-mail: mlogan{at}nimr.mrc.ac.uk
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Author contributions: C.M., J.J.G.-B., and M.P.L. designed research; C.M. and M.P.L. performed research; C.M. and M.P.L. analyzed data; and C.M., J.J.G.-B. and M.P.L. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. EU084005).
References
Tbx4/5 gene duplication and the origin of vertebrate paired appendages
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