Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression

Abstract

The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.[Keywords: cis regulation; Formin; global control region; Gremlin; limb development; regulatory landscape] Supplemental material is available at http://www.genesdev.org. Received February 8, 2004; revised version accepted May 5, 2004. The mouse is the genetic model of choice to study mammalian development and disease. In addition to alteration of specific genes by gene targeting and transgenesis, mutant mouse strains identified by phenotypic screens are commonly used to analyze developmental and disease processes (for reviews, see Justice 2000; Perkins 2002). A significant fraction of spontaneous mutations in mice (and humans) cause congenital limb malformations and have proven crucial to unravel the molecular mechanisms regulating vertebrate limb bud morphogenesis (for review, see Gurrieri et al. 2002). In particular, several alleles of the recessive mouse limb deformity (ld) mutation disrupt patterning of the distal limb skeleton. Over the years, a total of five ld alleles have been identified by phenotypic and genetic complementation analysis. All ld homozygous newborn mice display limb patterning defects characterized by synostosis of the zeugopod in combination with oligo-and syndactyly of metacarpal bones and digits (for review, see Zeller et al. 1999). In addition, ld homozygous newborn mice display varying degrees of uni-and bilateral renal aplasias depending on allele "strength" (Maas et al. 1994). Molecular analysis showed that the two ld alleles (ld

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