Analysis of the Function of Megf7 During Development

Lrp4/Megf7, a member of the low-density lipoprotein receptor gene family, is a major regulator of tissue patterning during embryonic development. Prior to this work, the function Megf7 had largely been unknown. Eight distinct mutations in Megf7 were introduced into mice in order to facilitate a functional analysis of Megf7. The Megf7EC Stop mutant, which has a premature stop codon inserted upstream of the transmembrane domain, had defects in limb, tooth, and mammary gland development. Subsequent analysis showed that the defects in limb development were caused by the expansion of the limb bud structure called the apical ectodermal ridge. Biochemical analysis suggests that Megf7 acts as an inhibitor of the Wnt signaling pathway both in vivo and in vitro. The analysis of the tooth defect of the Megf7EC Stop mutant shows that there is an increase in BMP activity, one of the major cellular signals involved in development. This increase in BMP activity leads to a loss of patterning during tooth development. The complete Megf7 null allele that was generated, in addition to having limbs defects, also failed to form kidneys and was paralyzed at birth. The kidney agenesis is the result of a failure of the ureteric bud to grow and branch during the initial stages of kidney development. The phenotype of the Megf7EC Stop and Megf7KO mutants prompted us to look for natural mutants in other animals. We found that Mulefoot Disease, a form of syndactyly in cows, is caused by a single base change at the exon/intron border of exon 37 of bovine Megf7. in vivo and in vitro data suggests that this mutation leads to altered splicing of the gene and premature truncation of the translated gene product. The other six mutant mouse alleles that were generated had specific mutations introduced into the cytoplasmic domain of Megf7. The limb phenotypes of these mice suggest that Megf7 may serve as an endocytic receptor. This work establishes Megf7 as a major regulator of patterning during development and is involved in a natural form of limb dysgenesis. This work will serve as the groundwork for future analysis of Megf7.