Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly

Abstract

Precise spatial and temporal control of developmental genes is crucial during embryogenesis. Regulatory mutations that cause the misexpression of key developmental genes may underlie a number of developmental abnormalities. The congenital abnormality preaxial polydactyly, extra digits, is an example of this novel class of mutations and is caused by ectopic expression of the signalling molecule Sonic Hedgehog (SHH) in the developing limb bud. Mutations in the long-distant, limb-specific cis-regulator for SHH, called the ZRS, are responsible for the ectopic expression which underlies the abnormality. Here, we show that populations of domestic cats which manifest extra digits, including the celebrated polydactylous Hemingway's cats, also contain mutations within the ZRS. The polydactylous cats add significantly to the number of mutations previously reported in mouse and human and to date, all are single nucleotide substitutions. A mouse transgenic assay shows that these single nucleotide substitutions operate as gain-of-function mutations that activate Shh expression at an ectopic embryonic site; and that the sequence context of the mutation is responsible for a variable regulatory output. The plasticity of the regulatory response correlates with both the phenotypic variability and with species differences. The polydactyly mutations define a new genetic mechanism that results in human congenital abnormalities and identifies a pathogenetic mechanism that may underlie other congenital diseases.

The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of Shh during limb development

“…Surprisingly, this effect was not confined to the ectopic anterior Shhexpressing region seen in the Slk leg, but was also found in the posterior Shh-expressing region of the Slk leg and wing. We, therefore, conclude that although misregulation of Shh occurs from the loss of a long-range Shh control element, as has been previously proposed (Lettice et al, , 2008, this is not an anterior specific element, but general to both the anterior and posterior regions of the limb, which have the potential to express Shh (Amano et al, 2009;Tanaka et al, 2000).…”

“…Mutations within the ZRS have been reported to induce ectopic anterior Shh expression in the developing mouse limb Lettice et al, 2003Lettice et al, , 2008 and it has been shown that Shh is expressed ectopically in the Slk leg (Arisawa et al, 2006). On examining Shh expression in the developing Slk leg, we observed that in addition to ectopic anterior expression previously reported (Arisawa et al, 2006), the posterior Shh domain was slightly larger in Slk embryos compared to wt legs (compare Fig.…”

“…This effect also occurs during limb development of mouse Hx mutants in which the posterior Shh expression domain is also much larger than in wt limbs (Blanc et al, 2002). Transgenic mouse experiments that have examined expression of lacZ in the limb driven by mutated versions of human, mouse, cat ZRS, and dog pZRS sequences, have found expression in the posterior of the limb is usually broader than with wt ZRS constructs (Lettice et al, 2008;Park et al, 2008). Thus, misregulation of Shh expression in the posterior of the Slk limb, due to a mutation in the ZRS, is not a chicken-specific effect.…”

“…Mutations in intron5 of LMBR1 are associated with ectopic Shh expression in the limb and/or polydactyly in humans, mice, and cats Masuya et al, 2007;Wang et al, 2007;Gurnett et al, 2007;Lettice et al, 2008). This intronic region has been termed the ''zone of polarising activity regulatory sequence'' (ZRS) see Supp.…”

“…Genetic evidence from mouse has shown that the ZRS sequence acts in cis with Shh (Lettice et al, 2002), physically interacting with the Shh sequence specifically in limb bud tissue to regulate Shh expression (Amano et al, 2009). Transgenic mice expressing lacZ under the control of human, mouse, cat, dog, or Fugu ZRS or pZRS sequences can replicate a posteriorly restricted expression pattern in the limb, similar to that of Shh (Lettice et al, , 2008Park et al, 2008;Maas and Fallon, 2005) whereas ZRS sequences containing mutations equivalent to those found in human, mouse, or cat polydactylous syndromes can also drive lacZ expression in the anterior of limbs in transgenic mice, mimicking the ectopic Shh seen in these polydactylous conditions (Lettice et al, , 2008Maas and Fallon, 2005). Thus, the ZRS sequence is not only important in the normal expression of Shh in the limb, but also plays a role in the induction of polydactyly in many species including human.…”