Lmx1b‐Mediated Emx2‐ Associated Regulatory Region Active During Limb Development

Lmx1b is a LIM-homeodomain transcription factor that has dorsally restricted expression in limb mesoderm and is responsible for limb dorsalization. Mice lacking functional  Lmx1b  exhibit ventral-ventral limb symmetry. The mechanisms used by Lmx1b to orchestrate limb dorsalization are unknown. A possible target is the transcription factor Emx2, which is upregulated 5.3 fold in the presence of Lmx1b during limb development. Furthermore, chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) identified bound Lmx1b within a 500 bp conserved noncoding region (CNR) 550 kb downstream of the  Emx2  coding sequence. We hypothesized that this  Emx2 -associated CNR would be active in limb development and Lmx1b-dependent. To test its activity, we generated a reporter construct containing the  Emx2 -associated CNR and electroporated it into embryonic chicks. To confirm Lmx1b's role, we repeated the experiment using constructs containing site-directed mutation of the Lmx1b binding site. Our results demonstrate enhancer activity within both the dorsal and ventral limb mesoderm during development. This activity is abolished if the Lmx1b-binding site is mutated. Thus, we have identified an  Emx2 -associated regulatory region ( EARR ) that binds to Lmx1b during limb development. Although  EARR  activity was not restricted to the Lmx1b expression domain, it was dependent on the Lmx1b binding site. This indicates that another transcription factor present in the ventral mesoderm can use the Lmx1b binding site. We suspect that in its normal genetic context, differential chromatin conformation may limit  EARR  activity to the Emx2 expression domain within the dorsal limb mesoderm rendering it Lmx1b-dependent.