Several bone morphogenetic proteins (BMPs) are expressed in the apical ectodermal ridge (AER), a critical signaling center that directs the outgrowth and patterning of limb mesoderm, but little is known about their function. To study the functions of apical ectodermal BMPs, an AER-specific promoter element from the Msx2 gene was used to target expression of the potent BMP antagonist noggin to the apical ectoderm of the limbs of transgenic mice. Msx2-noggin mutant mice have severely malformed limbs characterized by syndactyly, postaxial polydactyly, and dorsal transformations of ventral structures indicated by absence of ventral footpads and presence of supernumerary ventral nails. Mutant limb buds exhibit a dorsoventral (DV) and anteroposterior (AP) expansion in the extent of the AER. AER activity persists longer than normal and is maintained in regions of the apical ectoderm where its activity normally ceases. Mutant limbs possess a broad band of mesodermal tissue along the distal periphery that is absent from normal limbs and which fails to undergo the apoptosis that normally occurs in the subectodermal mesoderm. Taken together, our results suggest that apical ectodermal BMPs may delimit the boundaries of the AER by preventing adjacent nonridge ectodermal cells from becoming AER cells; negatively modulate AER activity and thus fine-tune the strength of AER signaling; and regulate the apoptosis of the distal subectodermal mesoderm that occurs as AER activity attenuates, an event that is essential for normal limb development. Our results also confirm that ectodermal BMP signaling regulates DV patterning.

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Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.

Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the apical ectodermal ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme. Studies of the altered expression patterns of Msx-2 in amelic and polydactylous mutant chick limbs have suggested that the apical ectodermal ridge and mesodermal domains of Msx-2 expression are independently regulated and that there might be separate cis-regulatory elements in the Msx-2 gene controlling its spatially distinct domains of expression. To test this hypothesis, we have isolated the chicken Msx-2 gene and have tested the ability of various regions of the gene to target expression of LacZ reporter gene to specific regions of the limbs of transgenic mice. A variety of these constructs are consistently expressed only in the apical ectodermal ridge and the ectoderm of the genital tubercle and are not expressed in the mesoderm of the limb bud or in other regions of the embryo where the endogenous Msx-2 gene is expressed. These results suggest the presence of spatially specific cis-regulatory elements in the Msx-2 gene. We identified a 348-bp region in the 5' flanking region of the Msx-2 gene which can act as an apical ectodermal ridge enhancer element when placed in reverse orientation in front of the reporter gene with transcription initiation directed by the minimal hsp68 promoter.