Twist Plays an Essential Role in FGF and SHH Signal Transduction during Mouse Limb Development

Abstract

Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist(-/-) forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signalling in the limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist(-/-) embryos is also retarded. The overall activity of genes involved in SHH signalling is reduced.Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signalling are expressed in normal patterns. Twist(+/-);Gli3(+/XtJ) mice display more severe polydactyly than that seen in either Twist(+/-) or Gli3(+/XtJ) mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction.

Preaxial polydactyly: interactions among ETV, TWIST1 and HAND2 control anterior-posterior patterning of the limb

“…In Twist1 heterozygous mutants, Shh expression is not only present in the ZPA, but is also detected in the anterior mesenchyme of the hindlimb bud (O'Rourke et al, 2002). However, in Twist1 homozygous mutants, Shh expression is diminished in the entire limb bud (O'Rourke et al, 2002;Zuniga et al, 2002).…”

“…Furthermore, Fgf10, a gene essential for AER formation, remained expressed in the mesenchyme of the Prx1cre;Twist1 mutant limb bud (see Fig. S1G,H in the supplementary material), in contrast to its downregulation in Twist1-null limb buds (O'Rourke et al, 2002;Zuniga et al, 2002). These results suggest that the transient presence of TWIST1 at the beginning of limb bud initiation is sufficient to maintain the expression of key genes in the limb bud mesenchyme, which in turn allows for formation and maintenance of the AER in this conditional mutant.…”

“…Xt-J/+ , a heterozygous mutant carrying a loss-of-function allele of Gli3, offers a sensitive genetic background as it has been shown to genetically interact with several other mutants that exhibit PPD phenotypes (Dunn et al, 1997;O'Rourke et al, 2002;Panman et al, 2005 Xt-J/+ single mutants. This result therefore distinguishes Twist1 from Gli3 as a specific Etv cooperator.…”

“…Twist1 heterozygous mutants exhibit hindlimb-specific PPD and ectopic Shh expression, suggesting that TWIST1 inhibits Shh expression (Bourgeois et al, 1998). However, Twist1 homozygous limb buds show overall diminished Shh expression, suggesting that TWIST1 is required for Shh expression (O'Rourke et al, 2002;Zuniga et al, 2002). This downregulation of Shh could be due to disruption of the apical ectodermal ridge (AER) observed in Twist1 homozygous limb buds.…”

Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois

“…5C,D). The twist gene, which is required in limb mesenchyme for the FGFmediated feedback loop and for proper FGF receptor expression in the limb mesenchyme (O'Rourke et al, 2002;Zuniga et al, 2002), is also expressed normally in Tbx4 mutants (Fig. 5E,F).…”