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GDF5 Is a Direct Target of LMX1B Regulation During Limb Dorsalization
Author(s) -
Pira Charmaine U,
Haro Endika,
Pernu Lauren M,
Casiano Carlos M,
Kim Emily J,
Aka Chelsea A,
Oberg Kerby C
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb34
Subject(s) - enhancer , biology , limb development , sox9 , homeobox , ectopic expression , transcription factor , gene , limb bud , regulation of gene expression , microbiology and biotechnology , genetics , anatomy
Lmx1b is a homeodomain transcription factor that is expressed in the dorsal mesoderm and plays a critical role in limb dorsalization. The absence of functional Lmx1b results in a ventral‐ventral limb phenotype in mice whereas ectopic Lmx1b expression causes dorsal‐dorsal limb morphology. Lmx1b‐regulated genes have been identified in developing limb buds by gene array analysis; however, direct targets of Lmx1b have yet to be confirmed. Lmx1b ChIP‐seq analysis of E12.5 mouse embryo limb tissue identified an Lmx1b bound interval (LBI) located 78Kb upstream of the Gdf5 gene, which is involved in joint development and structure and previously shown to be upregulated by Lmx1b at this stage of development. The LBI is highly conserved across divergent species and is associated with active chromatin regulatory marks supporting a role as a regulatory sequence. We isolated the LBI, cloned it into a GFP reporter construct and electroporated the construct into developing chick wings. We found that the LBI exhibited enhancer activity in limb joints, coincident with Gdf5 expression. Our findings support a recent report that identifies this cis ‐regulatory module (CRM) as regulator of Gdf5 expression in distal joints. In addition, using chromosome conformation capture (3C) technology, we found that this CRM interacts directly with the Gdf5 promoter. Collectively, these data indicate that Lmx1b directly regulates Gdf5 via a joint‐restricted CRM during limb dorsalization. Further studies are warranted to characterize this Gdf5 ‐associated enhancer and the role Lmx1b and its partners play in regulating Gdf5 expression during limb dorsalization.