Abstract
To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.
Highlights
Bmp4, a member of the TGF-b superfamily, is a secreted signaling molecule essential for embryogenesis [1,2,3]
We show here that Pitx homeoprotein family members bind a specific site in the Bmp4 incisor epithelium limb bud (‘‘incisor epithelium/limb bud’ (IE/LB)’’) enhancer that is necessary for its activity in vivo
We identified a 396 bp minimal ‘incisor epithelium/limb bud’ (IE/LB) Bmp4 enhancer that contains putative binding sites for members of the Pitx and Msx homeoprotein families, which are expressed with Bmp4 in these developing tissues
Summary
A member of the TGF-b superfamily, is a secreted signaling molecule essential for embryogenesis [1,2,3]. It is expressed in a variety of tissues and organs throughout embryonic and postnatal life [4]. Conditional deletion of Bmp in distal mandibular arch ectoderm results in mandibular truncation and lack of incisors, indicating essential functions of epithelial Bmp in these ectodermal organs [7]. Bmp has been implicated as one of the earliest signaling molecules secreted from the oral ectoderm that is capable of inducing dental mesenchymal genes that are necessary for tooth formation [7,11,12,13]
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