Elimination of a long-range cis-regulatory module causes complete loss of limb-specificShhexpression and truncation of the mouse limb

Abstract

Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog(Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants,ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range,cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.

Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms

“…That epigenetic changes can control development and disease is well accepted (see recent reviews: Rajasekhar and Begemann, 2007;Soshnikova and Duboule, 2008); therefore, it is reasonable to believe that epigenetic mechanisms will also play a role in regeneration. Yakushiji et al (2007) studied the methylation level of the enhancer region of a limb-specific shh enhancer, which has been conserved from fish to mammals (Sagai et al, 2005), in amphibians with different regenerative abilities. In Xenopus hindlimbs that regenerate and express shh, the enhancer was methylated on approximately half the CpG sites (40 -50%) in either developing or regenerating limbs, while in the pattern-deficient regenerating froglet forelimb, it was hypermethylated (84 -91%).…”

Distant regulatory elements in a Sox10‐βGEO BAC transgene are required for expression of Sox10 in the enteric nervous system and other neural crest‐derived tissues

“…A common theme is emerging that genes that are elaborately regulated during development are often controlled by modular cis-acting elements at a great distance from the start site of transcription. Studies in transgenic mice have revealed a spectrum of regulatory strategies that may be in effect: distant and scattered over a large interval (Biben et al, 1996;Moore et al, 1998;Antes et al, 2001;Mortlock et al, 2003), far 3Ј of the gene (DiLeone et al, 1998;Pape et al, 1999), intronic (Rowntree et al, 2001), or even embedded in neighboring genes (Carvajal et al, 2001;Sagai et al, 2005). Attempts to identify gene regulatory regions can be speeded significantly by use of bioinformatics tools to locate evolutionarily conserved noncoding sequences among species.…”

Remote Control of Gene Expression

“…Several proximal regulatory element knockouts have been generated in mice including the Hif1␣ response element of VEGF-A (38) and a CArG box upstream of the Telokin gene (39). A distal CRM of Shh, located nearly 1 megabase away from its promoter, has also been deleted in its native genomic context and shown to be essential for normal Shh expression (40). On the other hand, there have been some regulatory element knockouts with unpredictable outcomes.…”