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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01390

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A phylogenetically conserved cis-regulatory module in the Msx2 promoter is sufficient for BMP-dependent transcription in murine and Drosophila embryos

¹ Department of Biochemistry and Molecular Biology, Norris Cancer Hospital, USC Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA
² Department of Developmental and Cell Biology, Developmental Biology Center, UC Irvine, Irvine, CA 92697-2275, USA
³ Departments of Orthopedic Surgery and Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095-1737, USA
⁴ Department of Biology, California State University at Chico, Chico, CA 95929, USA

View larger version (67K): [in a new window]   Fig. 1. A 560 bp fragment of the Msx2 promoter is sufficient for BMP responsiveness in 10T1/2 cells and murine embryonic limbs. (A) Genomic map showing fragments of murine Msx2 5' flanking sequence used in transgenic and transfection experiments. (B) Results of transient transfections of 10T1/2 cells with Msx2 promoter-TK-luciferase vectors bearing either the 1.8 kb or 560 bp fragment. (C-F) Comparisons of endogenous Msx2 expression by whole-mount in situ hybridization (C,F) and Msx2 promoter-hsplacZ transgene expression (D,E). (G) Limbs derived from E11.5 560bpMsx2-hsplacZ transgenic embryos implanted with Affigel agarose beads soaked in BMP4, TGFß1 or BSA. Limbs were stained for ß-galactosidase activity for the indicated times. Anterior towards the right. The beads are light blue; a positive response to ligand produces dark-blue staining around the bead.

View larger version (82K): [in a new window]   Fig. 2. The Msx2 BMP-responsive region is highly conserved among mammals. BLAST was used to search for sequence identity between the murine Msx2 locus and Msx2 loci of other vertebrates. Alignments of a 480 bp homology block are shown in different mammalian species, with coordinates of transcription start sites at left. Sequence differences relative to chimpanzee are in bold. The underlined sequence (164-246) is a tandem duplication; bases in blue are those that differ between the two duplicated copies. Shaded areas show consensus binding sites for the indicated transcription factors. Boundaries of transgenes are indicated by brackets below the sequence. Putative Smad-binding sites within the 52 bp transgene are underlined. The boxed area indicates a putative Fox/Hnf site matching the consensus (C/A)(T/C)(C/A)AA(T/C)A in 5/7 positions.

View larger version (65K): [in a new window]   Fig. 3. Deletion analysis defines a 220 bp minimal Msx2 element and a 52 bp element required for BMP signaling. (A) Deletion series of lacZ constructs bearing fragments of the 560 bp promoter sequence. The BMP responsiveness of each transgene was assessed by bead implantation; positive response (+), negative response (–). (B,C,E,F,H) lacZ stains of 560 and 220 bp transgenic embryos at E11.5. Expression patterns (B,C) and BMP responsiveness (E,F) are similar. BMP-responsive constructs shared a 52 bp region (A, *), deletion of which in the context of the 560 bp transgene resulted in reduced expression (D), and loss of BMP responsiveness in embryos (G) and in 10T1/2 cells (I). Because of diminished transgene activity, the embryo in D was stained overnight (compare with H), while embryos in B and C were stained for 1 (B) and 2 (C) hours.

View larger version (73K): [in a new window]   Fig. 4. The 52 bp element responds to BMP signaling in a subset of sites relative to the parental 560 bp fragment. Expression and BMP-responsiveness of the 52bpMsx2-hsplacZ transgene were assessed in a developmental series of embryos and tissues from E8.5 to E13.5. A total of five transgenic lines were compared, with closely similar results (Table 1). (A-D) Overnight lacZ stains of embryo whole mounts. (E,F) Midsagittal sections of whole mounts stained for lacZ. The embryo in E was embedded in plastic and photographed in dark field, showing lacZ activity in pink. Note expression in the allantois (A,B), pharyngeal arches (A-C,E,F) and cardiac outflow tract (A-F). Expression in the pharyngeal region was restricted to tissues adjacent to the pharyngeal endoderm and cardiac outflow tract (E,F). At E11.5, expression persisted in the outflow tract, and was evident in the genital region, the anterior limb bud mesenchyme, the otic vesicle and the eye (D). (G-I) High-magnification views of forelimbs of E12.5-E13.5 embryos stained for lacZ (anterior to right). (G) Expression of the parental 560 bp transgene. (H,I) Expression of the 52 bp transgene in the anterior and interdigital limb mesenchyme. (J,K) lacZ expression in bead-implanted limbs. a, allantois; pam, pharyngeal arch mesenchyme; ot, outflow tract; al, anterior limb; gr, genital region; ov, otic vesicle; e, eye.

View larger version (63K): [in a new window]   Fig. 6. Smad and homeodomain consensus sites are necessary for BMP responsiveness of Msx2 transgenes. (A) Smad or homeodomain consensus sites (bold) were mutated in the 52 bp element as shown above and below the sequence (red). Potential Brinker (blue) and Smad4 (green) sites are identified. (B-D) EMSA was used to assess the effects of the mutations on binding of bacterially expressed Smad4 MH1 domain (B), and in vitro transcribed/translated Prx1b, a paired-class homeoprotein (D). Amounts of GST-Smad4 protein used were 50, 250 and 500ng (wild-type control; 500 ng GST). The relative amounts of Prx1b lysate used were 1, 2, 4 and 10 µl (controls; 20 µl). Band intensities of the faster-migrating, primary Smad4-DNA complexes are plotted in C. (E-J) Effect of Smad site and homeodomain site mutations on 52bpMsx2-hsplacZ transgene expression in transgenic embryos at E11.5 (E-G), and BMP responsiveness in limbs (H-J). (K-O) We introduced the homeodomain site mutation into the 560 bp fragment (Fig. 1A, Fig. 3A) and examined the expression and BMP responsiveness of this construct in 10T1/2 cells (K) and in transgenic embryos (L-O) at E11.5. Embryos in E-G,M were stained for lacZ activity overnight, while that in L was stained for 1 hour.

View larger version (61K): [in a new window]   Fig. 5. Phosphorylated forms of BMP-dependent R-Smads colocalize with 52 bp lacZ transgene expression in the limb and cardiac outflow tract, and are recruited to the Msx2 BMP-responsive region in native chromatin. Beads soaked in BMP4 were implanted on limbs of E11.5, 52bpMsx2-hsplacZ transgenic embryos. (A) Whole-mount lacZ stain showing expression and BMP response in the anterior limb (arrow). (B-E) Adjacent frozen sections along the dorsoventral axis of the limbs were either stained for lacZ activity (B,C; blue against Nuclear Fast Red counterstain), or immunostained with an antibody against the phosphorylated forms of the BMP R-Smads 1, 5 and 8 (D,E; pink against DAPI counterstain). (F-J) lacZ (G,I) and phospho-Smad (H,J) staining of adjacent midsagittal sections through cardiac region of a 52bpMsx2-hsplacZ embryo at E9.5 (F). A subset of phospho-Smad stained cells are positive for lacZ activity. (K-M) ChIP assay showing association of phosphorylated R-Smads with the Msx2 BMPRE in C14 limb mesenchymal cells. (K) Autoradiogram of RNA derived from BMP2-treated C14 cells, probed for Msx2. Upstream control and BMPRE primers (L) were used to interrogate chromatin immunoprecitated with anti phospo-Smad1 antibody (M).

View larger version (63K): [in a new window]   Fig. 7. The Msx2 BMPRE accurately interprets Dpp signals in Drosophila. (A-D) Msx2 transgenes behave like Dpp-responsive genes in imaginal discs. In situ hybridization showing dpp expression (A). Expression of a vestigial-lacZ reporter gene, a known dpp target (B). Expression of the 480bpMsx2-lacZ transgene (C). The 480 bp fragment is the conserved region identified in Fig. 2, Fig. 3A. Expression of the 480bpMsx2-lacZ transgene in the wing pouch with ectopic activation of the Dpp pathway by A9Gal4>TkvA (D). (E-I) dpp and 220bpMsx2-lacZ transgene expression in stage 13 embryos (lateral view). dpp in situ hybridization showing wild-type expression pattern in parasegments (ps) 3 and 7 of the embryonic mid-gut (E) and in dpp (S11/S22) mutant (F), in which dpp expression is lost in ps3 (arrow), but not in ps7. 220bpMsx2-lacZ transgene in wild type (G) and dpp (S11/S22) mutant (H). Expression of 220bpMsx2-lacZ transgene throughout the gut in response to ectopic expression of Dpp driven by a heat-shock promoter (I). (J-Q) dpp and 52bpMsx2-lacZ transgene expression in early blastoderm embryos (lateral view). dpp in situ of a stage 5 embryo showing restriction of dpp expression to the dorsal part of the embryo (J). Expression of the 52bpMsx2-lacZ transgene in wild-type (K) and dorsal mutant (L) embryos. Expression of 52bpMsx2-lacZ transgene in screw (M) and brinker (O) mutant embryos, and in an embryo with ectopic activation of Dpp using Tub Gal4>UAS Dpp (N). 52bpMsx2-lacZ transgene expression is lost when the Smad sites (P) or homeodomain site (Q) are mutated.

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