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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01327

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HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis

Wendy M. Knosp^2,*, Virginia Scott^1,*, Hans Peter Bächinger^1,3 and H. Scott Stadler^1,2,

¹ Shriners Hospital for Children, Research Division, Portland, Oregon 97239, USA
² Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon 97239, USA
³ Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA

View larger version (101K): [in a new window]   Fig. 1. Spatiotemporal expression of Hoxa13 correlates with sites of malformation and decreased BMP expression. (A-D) Analysis of Hoxa13 expression by in situ hybridization using an exon 1-specific riboprobe. Hoxa13 expression is present in the distal interdigital mesenchyme and peridigital tissues (A, arrowheads). (C) Hoxa13 localizes to the distal joint/nail bed in E14.5 limbs (arrowheads). (B,D) In homozygous mutants, elevated levels of Hoxa13 exon 1 transcripts were detected throughout the autopod. (E,F) Alcian Blue staining of E15.5 mutant limbs reveals defects in distal digit separation (F, arrows) and chondrogenesis (arrowheads), when compared with wild-type controls. Pi, pisiform carpal element. (G-J) Bmp7 expression is reduced (arrows) in the distal interdigital tissues at E12.5 (G,H) and in the peridigital tissues at E13.5 (I,J) of homozygous Hoxa13 mutants, when compared with age-matched wild-type controls (arrowheads). (K-N) Bmp2 expression is reduced (arrows) in the interdigital tissues of E12.5 Hoxa13 mutant embryos, and in the distal joints/nail beds of E14.5 Hoxa13 mutants, when compared with wild-type controls (arrowheads). (O-R) Msx2, a target of BMP signaling, exhibits reduced interdigital expression in E12.5-E13.5 mutant limbs (arrows) compared with age-matched controls (arrowheads). (S,U) BMP7 (red, arrowhead) and HOXA13-GFP (green) co-localize (yellow cells in U and V) in the interdigital tissues of E12.5 limbs. (T,V) Age-matched homozygous mutants exhibit reduced numbers of BMP7-positive cells in the same interdigital regions (arrow), a finding consistent with the reduced Bmp7 transcripts in these same tissues (compare H and T). Scale bar: 50 µm.

View larger version (46K): [in a new window]   Fig. 2. Structural and functional analysis of the HOXA13 DNA-binding domain. (A) The amino acid sequence of the A13-DBD peptide is presented above. (Left panel) Structural analysis of the A13-DBD peptide by circular dichroism spectroscopy indicates that the peptide folds into a stable -helical DNA-binding motif at 4°C (blue curve). At 60°C, the majority of the -helical content is lost by thermal denaturation (red curve). (Right panel) Thermal stability measurements of the A13-DBD peptide showed that the -helical conformation is maintained between 4 and 25°C. At temperatures higher than 25°C, the peptide cooperatively transforms to its denatured conformation. (B-E) A13-DBD peptide exhibits specific binding for the DNA regions present upstream of Bmp2 and Bmp7. Asterisks denote unbound radiolabeled DNA, arrowheads denote A13-DBD-DNA complexes (B,C,E). (B) Quantitation of the A13-DBD affinity for the bound DNA regions using increasing concentrations (black triangles) of cold competitor DNA (0, 150, 300 and 750 nM) revealed differential A13-DBD affinities for each of the bound sites. (C) The Bmp2C2 region requires 2-fold greater concentrations of A13-DBD to affect its electrophoretic mobility. (D) Radiolabeled control DNA sequences exhibited no change in electrophoretic mobility when incubated with 4-fold (2 µM) higher concentrations of the A13-DBD peptide. (E) A13-DBD binding specificity was confirmed using the Bmp7C1 binding site, which could not be displaced from the A13-DBD peptide by using 750 nM concentrations of the unlabeled negative control DNA (cNC), but is completely displaced using 750 nM unlabeled Bmp7C1 competitor DNA (cBMP7C1). (F) Analysis of the DNA sequences bound by the A13-DBD reveals a novel series of HOX binding sites. The core TAAT site is designated in bold type.

View larger version (62K): [in a new window]   Fig. 3. HOXA13 activates transcription from the Bmp2 and Bmp7 enhancer regions, and associates with these enhancers in vivo. Co-transfection of NG108-15 cells with luciferase reporter plasmids containing forward or reverse orientations of the Bmp2 enhancer sequence (A,C), or the Bmp7 enhancer sequence (B,C), and pCMV-A13 resulted in 2.5- and 1.8-fold (Bmp2), and 1.8- and 2.0-fold (Bmp7), increases in RLA, when compared with identical transfections with the control pCMV vector. Luciferase activity was normalized for transfection efficiency using a Renilla Luciferase control plasmid in all co-transfection assays. Bars represent the standard deviation of results derived from four transfection assays. (D) Western blot analysis of protein lysates derived from Hoxa13 wild-type (+/+), heterozygous mutant (+/–) and homozygous mutant (–/–) tissues confirms that the Hoxa13 antibody recognizes proteins of the correct molecular weight for wild-type HOXA13 (43 kDa) and mutant HOXA13-GFP (64 kDa). (E) The Hoxa13 antibody immunoprecipitates HA-tagged full-length HOXA13. (F-I) Immunostaining of cultured limb mesenchyme from HOXA13-GFP mutant mice, using the Hoxa13 antibody, reveals strong nuclear co-localization (H, yellow, arrows) between the endogenous HOXA13-GFP protein (F) and the Hoxa13 antibody (G,H). Nuclei are stained with DAPI (I). (J-L) Chromatin immunoprecipitation using the Hoxa13 antibody confirms that wild-type (+/+) HOXA13 binds the Bmp2 (J) and Bmp7s1 (K) enhancer regions in the developing limb, whereas immunoprecipitates from mutant limbs (–/–) lacking the HOXA13 DNA-binding domain did not contain the Bmp2 and Bmp7s1 enhancer regions. (L) The absence of Bmp7s2 sequences in wild-type immunoprecipitates confirms HOXA13 specificity for the TAAT-containing sequences in Bmp2 and Bmp7s1. The TAAT-containing sequences present in the Bmp2, Bmp7s1 and Bmp7s2 regions are listed below panels J, K, and L. NC, negative PCR control; PC, positive PCR control.

View larger version (72K): [in a new window]   Fig. 4. Supplementation of Hoxa13 homozygous mutant limbs with BMP2 or BMP7 partially restores interdigital programmed cell death (IPCD) and Msx2 expression. (A-C) Hoxa13 heterozygotes exhibited elevated levels of IPCD when treated with beads soaked in 0.1 mg/ml rhBMP2 or rhBMP7, whereas PBS control beads did not elevate IPCD beyond normal levels in organ cultured limbs. (D-F) IPCD is partially restored in Hoxa13 homozygous mutants in the presence of rhBMP2- or rhBMP7-treated beads, whereas PBS-treated beads did not re-initiate IPCD. Arrows denote sites of IPCD as detected by TUNEL assay (red signal). (G-J) Organ cultures of Hoxa13 mutant limbs treated with rhBMP2 or rhBMP7 exhibit reduced levels of Msx2 induction (arrows), when compared with age-matched controls. (K,L) Control treatments using PBS-beads had no affect on Msx2 expression (arrows). Roman numerals denote digits II, III and IV. Scale bar: 50 µm.

View larger version (69K): [in a new window]   Fig. 5. IPCD is delayed in Bmp7 homozygous mutant limbs. (A,B) TUNEL analysis of IPCD in E13.5 Bmp7 mutants (B) revealed a delay in IPCD between digits II and III (arrow), when compared with age-matched heterozygous controls (A, arrowhead). (C,D) Bright-field analysis of the same Bmp7 heterozygous and mutant limbs shown in A and B. (E-G) Analysis of Hoxa13 induction by exogenous BMP2 or BMP7. Wild-type limbs treated with 0.1 mg/ml BMP2 (E) or BMP7 (F) did not exhibit any increase in Hoxa13 expression when compared with PBS controls (G), indicating that in the developing autopod, BMP2 and BMP7 do not regulate Hoxa13 expression.