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T-box gene tbx5 is essential for formation of the pectoral limb bud

Abstract

The T-box genes Tbx4 and Tbx5 have been shown to have key functions in the specification of the identity of the vertebrate forelimb (Tbx5) and hindlimb (Tbx4)1,2. Here we show that in zebrafish, Tbx5 has an additional early function that precedes the formation of the limb bud itself. Functional knockdown of zebrafish tbx5 through the use of an antisense oligonucleotide resulted in a failure to initiate fin bud formation, leading to the complete loss of pectoral fins. The function of the tbx5 gene in the development of zebrafish forelimbs seems to involve the directed migration of individual lateral-plate mesodermal cells into the future limb-bud-producing region. The primary defect seen in the tbx5-knockdown phenotype is similar to the primary defects described in known T-box-gene mutants such as the spadetail mutant of zebrafish3,4 and the Brachyury mutant of the mouse5, which both similarly exhibit an altered migration of mesodermal cells. A common function for many of the T-box genes might therefore be in mediating the proper migration and/or changes in adhesive properties of early embryonic cells.

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Figure 1: Defects in pectoral fin formation in zebrafish embryos injected with anti-tbx5 morpholino oligonucleotides.
Figure 2: Expression of marker genes in embryos injected with the control oligonucleotide (a, c, e, g) (5 ng per embryo) and an anti-tbx5 morpholino oligonucleotide (b, d, f, h) (5 ng per embryo).
Figure 3: Expression of tbx5 in embryos injected with the control oligonucleotide (a, c, e, g) (5 ng per embryo) and an anti-tbx5 morpholino oligonucleotide (b, d, f, h) (5 ng per embryo).
Figure 4: Rescued formation of a pectoral fin bud by transplanted wild-type cells in a tbx5 morpholino-injected embryo.
Figure 5: Reduction in pectoral fin size and defects in cartilage formation in 5-day-old larval zebrafish injected with low concentrations of anti-tbx5 morpholino oligonucleotide (1–3 ng per embryo).

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Acknowledgements

We thank A. Oates for helpful suggestions for the construction of the test construct for morpholino oligonucleotides, A. Bruce, A. Oates and I. Skromne for critical comments on the manuscript, I. Ruvinsky for helpful discussions, and H. Dow for fish care. This work was supported by a postdoctoral fellowship from the American Heart Association, Heritage Affiliate (to D.A.), a Cancer Training Grant from NIH (to M.J.K.), and grants from NIH (to L.M.S. and R.K.H.) and NSF (to R.K.H.).

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Ahn, Dg., Kourakis, M., Rohde, L. et al. T-box gene tbx5 is essential for formation of the pectoral limb bud. Nature 417, 754–758 (2002). https://doi.org/10.1038/nature00814

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