Emx2 Regulation by Lmx1b

The tetrapod limb develops asymmetrically along several axes. Lmx1b is responsible for dorsalization of the distal limb; knockout (KO) mice show ventral‐ventral limb patterning and reduction of the scapula, a dorsal structure of the limb girdle. However, the mechanism and downstream targets of Lmx1b are still unknown. We have previously identified by gene array that Emx2 is down‐regulated in Lmx1b KO limbs. Emx2 has been implicated in scapular development and Emx2 KO mice have scapular agenesis. We have identified three potential Lmx1b binding sites within the full length Emx2 gene (in the 5′UTR, in the second intron, and in the 3′UTR). We propose that these three sites are important for Lmx1b regulation of Emx2 . To determine the effect of Lmx1b on the expression of Emx2 , 293 HEK cells were co‐transfected with a full length genomic Emx2 vector in the presence of either an Lmx1b expression plasmid or Lmx1b siRNA. To determine the efficacy of the 5′UTR binding site, we fused the Emx2 5′UTR to GFP ( Emx2 ‐5′UTR‐GFP ) and examined reporter expression in the presence and absence of Lmx1b in 293 cells. Total RNA from transfected cells was extracted and RT‐PCR was performed to detect Emx2 and/or GFP expression. We found that Lmx1b upregulates Emx2 expression about 3‐fold over baseline expression. However, when the 5′UTR binding site is isolated and fused to GFP, Lmx1b inhibited reporter expression, whereas Lmx1b siRNA released Lmx1b inhibition. Deletion of the 5′UTR binding site resulted in a greater than 5‐fold increase in GFP expression, coupled with a loss of Lmx1b associated regulation. These data suggest that Lmx1b up‐regulates Emx2 expression, however, not by the 5′UTR binding site.