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Lmx1b‐Mediated Limb Dorsalization: Identification of a Potential Regulatory Sequence for Keratocan, Lumican, and Decorin
Author(s) -
Spady Rachael Nicole,
Haro Endika,
Pira Charmaine U,
Feenstra Jennifer M,
Oberg Kerby C
Publication year - 2016
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.30.1_supplement.1032.5
Subject(s) - biology , chromatin immunoprecipitation , mesoderm , lumican , enhancer , decorin , microbiology and biotechnology , genetics , limb bud , regulatory sequence , transcription factor , homeobox , reporter gene , limb development , gene , promoter , gene expression , proteoglycan , extracellular matrix , embryonic stem cell
Lmx1b is a homeodomain transcription factor expressed within the dorsal limb mesoderm. Lmx1b knock‐out mice develop ventral‐ventral limbs, while ventral overexpression results in dorsal‐dorsal limbs. Our goal is to further characterize Lmx1b‐regulated limb dorsalization by identifying downstream target genes. Gene array studies show up‐regulation of the extracellular matrix genes Keratocan (Ker), Lumican (Lum), and Decorin (Dcn) in the presence of Lmx1b. These three genes are clustered within 134k bp of each other on chromosome 11 in mice. Lmx1b‐targeted chromatin immunoprecipitation followed by massively parallel sequencing (Lmx1b‐ChIP‐seq) identified a potential regulator sequence (PRS), 509 bp in length, located 929 kb downstream of Lum with 3 potential Lmx1b binding sites. We reasoned that Lmx1b binding to this PRS might regulate Ker, Dcn and/or Lum expression during limb dorsalization. To determine activity, the PRS was isolated by PCR, incorporated into a construct with a basal promoter linked to GFP reporter, and then electroporated into the presumptive wing field of stage 14 chick embryos. We co‐transfected a β‐actin promoter‐driven RFP construct with the PRS reporter to confirm transfection efficiency. To determine the requirement for Lmx1b's binding within the PRS, we repeated the experiment following site directed mutagenesis of the predicted Lmx1b binding sites. The PRS is active in the ventral mesoderm of the developing limbs and absent in dorsal limb mesoderm proximally. This suggested that Lmx1b might inhibit activity of this PRS. However, there was no apparent change in enhancer activity after site‐directed mutagenesis of the 3 Lmx1b binding sites. Thus, Lmx1b is not likely to play a direct role in restricting this enhancer from dorsal mesoderm. Further analyses will be required to determine the factors responsible for this unique pattern of enhancer activity. Support or Funding Information Loma Linda University; National Organization of Rare Diseases (NORD)