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). 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 zebrafish and the Brachyury mutant of the mouse, 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.

Finger or toe: the molecular basis of limb identity

“…Consistent with this model, deletion of Tbx5 in the mouse results in the loss of Fgf10 expression in the limb bud mesenchyme (Agarwal et al, 2003;Rallis et al, 2003). Similarly, following knockdown of tbx5 function in zebrafish (Ahn et al, 2002;Ng et al, 2002), or misexpression of dominant-negative forms of Tbx5 in the chick (Ng et al, 2002;Takeuchi et al, 2003), Fgf10 expression is either absent or downregulated in the developing limb. As a consequence of failure of normal Fgf10 signalling in Tbx5 mutants, Fgf8 expression is also disrupted in the limb (Agarwal et al, 2003;Ng et al, 2002;Rallis et al, 2003;Takeuchi et al, 2003).…”

“…This indicates that tbx5 is required for the proper migration of cells that will form the pectoral fin (Ahn et al, 2002). It is unclear whether this observation has any significance in higher vertebrates because, in chick and mouse, Tbx5 is first detected in the region of the LPM that gives rise to the forelimb buds (Gibson-Brown et al, 1996;Gibson-Brown et al, 1998), and these cells do not undergo a similar migration.…”

“…To understand the role of Tbx5 in early limb formation, it is necessary to review our current understanding of the molecules involved in limb initiation. A number of experimental approaches in a range of model organisms indicate that Tbx5 is essential for forelimb bud formation, and that it interacts with the Fgf and Wnt signalling pathways to initiate limb bud outgrowth (Agarwal et al, 2003;Ahn et al, 2002;Ng et al, 2002;Rallis et al, 2003;Takeuchi et al, 2003). In the following sections, the current understanding of the interactions between Tbx5 and Fgf and Wnt family proteins, which initiate and maintain limb bud formation, is outlined.…”

“…Functional knockdown of tbx5 in the zebrafish using antisense morpholino oligonucleotides results in a failure to initiate pectoral fin bud formation, and the absence of fgf10 and fgf8 expression in the fin bud mesenchyme and AER, respectively (Ahn et al, 2002;Garrity et al, 2002;Ng et al, 2002). A mutation in the zebrafish tbx5 gene heartstrings, induced by the chemical mutagen ENU, produces an identical phenotype to the tbx5 morpholinoknockdown phenotype .…”

Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish

“…For each embryo, 1 nl of MO solution was injected into the cytoplasm. The sequence of MOs are: kctd10 ATG MO, 5 0 -TCT CTTCCATAGAAATTCCCCGATT-3 0 ; has2 MO, 5 0 -AGCAGCTCTTTGGAGA TGTCCCGTT-3 0 ; tbx5 MO, 5 0 -GAAA GGTGTCTTCACTGTCCGCCAT-3 0 (Gene Tools) 44,45 . Full-length capped zebrafish kctd10 mRNA was prepared using the SP6 mMessage mMachine kit (Ambion).…”