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

Abstract

SUMMARYPreaxial polydactyly (PPD) is a common limb-associated birth defect characterized by extra digit(s) in the anterior autopod. It often results from ectopic sonic hedgehog (Shh) expression in the anterior limb bud. Although several transcription factors are known to restrict Shh expression to the posterior limb bud, how they function together remains unclear. Here we provide evidence from mouse conditional knockout limb buds that the bHLH family transcription factor gene Twist1 is required to inhibit Shh expression in the anterior limb bud mesenchyme. More importantly, we uncovered genetic synergism between Twist1 and the ETS family transcription factor genes Etv4 and Etv5 (collectively Etv), which also inhibit Shh expression. Biochemical data suggest that this genetic interaction is a result of direct association between TWIST1 and ETV proteins. Previous studies have shown that TWIST1 functions by forming homodimers or heterodimers with other bHLH factors including HAND2, a key positive regulator of Shh expression. We found that the PPD phenotype observed in Etv mutants is suppressed by a mutation in Hand2, indicative of genetic antagonism. Furthermore, overexpression of ETV proteins influences the dimerization of these bHLH factors. Together, our data suggest that through biochemical interactions, the Shh expression regulators ETV, TWIST1 and HAND2 attain a precise balance to establish anterior-posterior patterning of the limb.

Msx1 and Msx2 in limb mesenchyme modulate digit number and identity

“…It should be noticed that, in the Twist1 null mutant, Etv4 and Etv5 are also up-regulated in the anterior limb bud mesenchyme, which does not preclude Shh derepression anteriorly. Indeed, it has been recently demonstrated that Etv and Twist1 proteins form a complex that is required to repress Shh anteriorly (Zhang et al, 2010). The similarity between these and our results may suggest a similar mechanism for anterior repression of Shh by Msx.…”

“…It should be noted that the Prx1-Cre transgene activity takes place progressively in both fore-and hindlimbs (Logan et al, 2002), and that, at the earliest stages of limb outgrowth, some Msx activity is likely to remain in the mesenchyme of our conditional mutant. A recent study (Zhang et al, 2010) has shown that for Twist1, a gene specifically expressed in the limb bud mesoderm, the Prx1-Cre-induced null mutation leads to a less dramatic phenotype than the constitutive null mutation. In particular, AER development is complete in the conditional null mutant and impaired in the constitutive one.…”

Persistent expression of Twist1 in chondrocytes causes growth plate abnormalities and dwarfism in mice

“…Sustained expression of Twist1 in cartilage led to a growth phenotype, characterized by shortening of the limbs and reduced body mass. Various Twist1 loss of function studies have also reported growth abnormalities and limb dismorphogenesis (Firulli et al, 2005, Firulli et al, 2007, Krawchuk et al, 2010, Zhang et al, 2010, thus demonstrating that in addition to its established function during intramembranous bone formation, Twist1 also plays a role in endochondral ossification.…”

“…Moreover, their histological analyses of the developing ribs and limbs in a1(II) Collagen-Cre; Twist1 fx embryos (E13.5 and E16.5), showed no obvious phenotype attributable to Twist1 gene inactivation in chondrocytes (Hinoi et al, 2006). However, conditional Twist1 abrogation from the limb mesenchyme by crossing Twist1 fx mice with mice containing the Prx1-Cre transgene, which activates Cre expression in prechondrocytes, resulted in forelimb patterning defects, hypoplastic scapula and clavicle, tibial aplasia, and preaxial polydactyly characterized by severe disorganization of cartilage elements (Krawchuk et al, 2010, Zhang et al, 2010. We speculated that the temporal requirement for Twist1 during postnatal chondrocyte formation may provide novel insight into stage-and dose-dependent roles of Twist1 during cartilage maturation and maintenance.…”

“…Moreover, Twist1 plays a role in immature chondrocytes of the perichondrium during skeletal development by regulating Runx2 activation of FGF18 expression (Hinoi et al, 2006). Two studies using several Twist1 mutant alleles demonstrated that incremental reduction of Twist1 activity during early limb bud patterning alters Shh expression domains and markedly affects limb and girdle development (Krawchuk et al, 2010,, Zhang et al, 2010. Removal of Twist1 activity from the limb mesenchyme using a floxed conditional null allele (Twist1 fx ) crossed with the Prx-Cre transgene resulted in forelimb patterning defects, hypoplastic scapula and clavicle, tibial aplasia and preaxial polydactyly exhibiting disorganized cartilage elements (Krawchuk et al, 2010).…”