Full size image available through contact |
The development of limb structures is regulated by molecular signalling cascades, some of which have been well defined to date. Similar signalling pathways are involved in outgrowth and patterning in both the fore- and hindlimbs. However, in the chick fore- and hindlimbs are also characterised by marked morphological differences. For example, while the forelimb (wing) develops three digits and is completely covered by feathers, the hindlimb (leg) develops 4 digits and is covered by scales and tuberculae.
Members of the T-box family have been identified in a variety of invertebrates and vertebrates, including humans. These genes encode transcription factors which are involved in regulation of gene transcription.
Tbx4 and Tbx5 genes are specifically expressed in the leg bud and wing bud respectively. Previous studies correlated these genes with specification of limb type identity. Toshihiko Ogura, Jun Takeuchi and coworkers (Nara Institute of Science and Technologie, Nara, Japan) in collaboration with Astrid Vogel-Höpker (Max Planck Institute for Brain Research, Frankfurt/Main, Germany, a recipient of a Japan Promotion of Science Fellowship ) have now shown that misexpression of Tbx4, a hindlimb specific gene, in the presumptive forelimb region of the chick embryo, induced the development of a leg-like structure at the position, where normally a forelimb would develop. The corresponding result, induction of forelimb-like structures in the hindlimb region was observed when the forelimb-specific gene Tbx5, was misexpressed in that region. The phenotypes of these misexpression studies were apparant both morphologically and on the basis of limb-specific expression of molecular markers (e.g. Hox genes). For example, misexpression of Tbx4 in the presumptive forelimb region induced the expression of hindlimb specific genes. In some cases Takeuchi et al could also induce the development of 4 digits and scale formation which are features characteristic for the hindlimb. Furthermore, the authors could show that Tbx4 is a downstream target of another hindlimb specific gene, Pitx1. These results published in Nature (29 April 1999) implicate these important genes in the determination of fore- and hindlimb identity in vertebrates.
Full size image available through contact |
In this study Takeuchi et al used an important new approach for studying limb development. In order to induce a precisely timed and locally restricted expression of Tbx4 or Tbx5, the researchers utilized a novel technology for incorporating genes into chick limb bud cells in vivo, referred to as micro-electroporation. This technique was first developed by Dr. Kunio Yasuda (Nara Institute of Science and Technology). Taking advantage of their large negative electrical charge, DNA molecules can be forced to move in an electrical field. Using ultrafine tungsten electrodes positioned at the desired target region, the applied retroviral construct carrying the gene of interest, penetrates through the cell membranes forced by the electric field. As a result a gene(s) can be quickly expressed at a place, where no expression is found during normal embryonic development. After several days of development, molecular and morphological analysis of the developing structures reveales insights into the functions of those genes during normal develpoment.
The results provide further insights into the genetic pathways related to human disease. In Holt-Oram-Syndrome, mutations in the Tbx5 gene causes severe malformations of the upper limbs (arms), further implicating T-box genes as important mediators in vertebrate pattern formation. With regard to evolution, the Tbx-subfamily of T-box genes are good candidates for key regulators of the development of paired appendages in all tetrapodes. The expression patterns strongly suggest that their function of regulating the fate of limb type is conserved from the pectoral and pelvic fins of ancestral fish to the arms and legs of humans. By providing the first functional demonstration in the model system of the developing chick embryo, this study has provided further experimental data to confirm and strengthen this hypothesis.
This work was supported by Special Coordination Funds for Promoting Science and Technology from Science and Technology Agency of Japan, Japan Society for the Promotion of Science Fellowship and the Deutsche Forschungsgemeinschaft.
Journal
Nature