Tbx4/5 gene duplication and the origin of vertebrate paired appendages

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.S ince 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 presump...

Regulation of limb bud initiation and limb‐type morphology

“…In transgenic analysis, constructs containing fragments of the mouse Tbx5 and Tbx4 genomic regions are able to drive gene expression in the forelimb-and hindlimbforming regions of the LPM while, in contrast, the amphiTbx4/5 genomic locus appears to lack the regulatory modules enabling expression in the LPM. In accordance with broadly accepted evolutionary models, these results suggest that changes at the level of the regulation of Tbx5 and Tbx4expression, rather than the generation of novel protein function, was necessary for the acquisition of paired appendages during vertebrate evolution (Horton et al, 2008;Minguillon et al, 2009). …”

“…Consistent with these observations, Pitx1 misexpression in the mouse forelimb results in transformation and translocation of specific muscles, tendons, and bones to acquire a hindlimb-like morphology (DeLaurier et al, 2006). Significantly, equivalent transgenic lines that express ectopic Tbx4 in the forelimb or Tbx5 in the hindlimb do not produce any alterations to limb-type morphologies again consistent with neither gene having a role in determining limb-type morphologies (Minguillon et al, 2005(Minguillon et al, , 2009). In Pitx1 null mice, the hindlimb skeleton loses some of its characteristic features (Lanctot et al, 1999;Szeto et al, 1999;Marcil et al, 2003).…”

“…Following conditional deletion of Tbx5 and simultaneous replacement with transgenically-supplied Tbx4, forelimb outgrowth and morphologies are rescued (Minguillon et al, 2005(Minguillon et al, , 2009). These results demonstrate that Tbx4 is sufficient to compensate for Tbx5 in forelimb initiation and that, in this context, it is not able to determine hindlimb morphologies.…”

“…In the background of the conditional deletion of Tbx5, an amphiTbx4/5 transgene is able to rescue forelimb formation. This demonstrates that the ancestral gene, present in limbless organisms, has the ability to initiate limb formation if expressed in the appropriate location (Minguillon et al, 2009). In transgenic analysis, constructs containing fragments of the mouse Tbx5 and Tbx4 genomic regions are able to drive gene expression in the forelimb-and hindlimbforming regions of the LPM while, in contrast, the amphiTbx4/5 genomic locus appears to lack the regulatory modules enabling expression in the LPM.…”

TBX5 intragenic duplication: a family with an atypical Holt–Oram syndrome phenotype

“…It is suggested essential cis-regulatory elements (located in flaking or intragenic regions) are likely to be found in close vicinity to these loci. 52 Minguillon et al 53 showed that changes at the regulatory level of Tbx4 and Tbx5 expression were necessary for the development of paired appendages during vertebrate evolution, rather than the generation of novel protein function. We speculate that the atypical HOS phenotype seen in this family could be due to a number of possible underlying pathogenetic mechanisms.…”