Anti-BARX2 monoclonal antibody, clone 9B8/2

Homeobox genes are regulators of place-dependent morphogenesis and play important roles in controlling the expression patterns of cell adhesion molecules (CAMs). To identify proteins that bind to a regulatory clement common to the genes for two neural CAMs, Ng-CAM and L1, we screened a mouse cDNA expression library with a concatamer of the sequence CCATTAGPyGA and found a new homeobox gene, which we have called Barx2, The homeodomain encoded by Barx2 is 87% identical to that of Barx1, and both genes are related to genes at the Bar locus of Drosophila melanogaster, Barx1 and Barx2 also encode an identical stretch of 17 residues downstream of the homeobox; otherwise, they share no appreciable homology. In vitro, Barx2 stimulated activity of an L1 promoter construct containing the CCATTAGPyGA motif but repressed activity when this sequence was deleted, Localization studies showed that expression of Barx1 and Barx2 overlap in the nervous system, particularly in the telencephalon, spinal cord, and dorsal root ganglia, Barx2 was also prominently expressed in the floor plate and in Rathke's pouch, During craniofacial development, Barx1 and Barx2 showed complementary patterns of expression: whereas Barx1 appeared in the mesenchyme of the mandibular and maxillary processes, Barx2 was observed in the ectodermal lining of these tissues, Intense expression of Barx2 was observed in small groups of cells undergoing tissue remodeling, such as ectodermal cells within indentations surrounding the eye and maxillo-nasal groove and in the first branchial pouch, lung buds, precartilagenous condensations, and mesenchyme of the limb, The localization data, combined with Barx2's dual function as activator and repressor, suggest that Barx2 may differentially control the expression of L1 and other target genes during embryonic development.

The downregulation of Pax7 expression is an essential requirement for adult muscle progenitor cell differentiation during muscle regeneration. We previously found that canonical Wnt signals drive myogenic differentiation by positively regulating the expression of myogenic target genes, and also by negatively regulating Pax7 expression. To better understand how Wnt signals repress Pax7 expression, we screened for Wnt-regulated microRNAs (miRNAs) that could target the Pax7 3 ' untranslated region (UTR). Using wild-type and beta-catenin null primary mouse myoblasts, we identified several Wnt/beta-catenin regulated miRNAs, the most abundant of which were miR-133b and miR-206. While miR-206 was previously identified as Pax7 regulator, miR-133b has never been shown to regulate the Pax7 transcript. We show here that miR-133b is a more potent inhibitor of Pax7 expression than miR-206, and that it acts via a site adjacent to the miR-206 binding site in the Pax7 3 ' UTR. The primary transcript encoding miR-133b/miR-206 is specifically induced by Wnt/beta-catenin, while the miR-1/miR-133a transcript is not; moreover, Wnt signals increase the secretion of mature miR-133b/miR-206 into exosomes. Overall, we conclude that miR-133b and to a lesser degree miR-206, but not miR-1 or miR-133a, are key components of the canonical Wnt-mediated pathway that allows differentiation to proceed by relieving Pax7-mediated repression of the myogenic program. Secretion of these miRNAs into exosomes may allow them to directly control the differentiation of neighboring cells.