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First published online 19 April 2006
doi: 10.1242/dev.02368

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Mutation of an upstream cleavage site in the BMP4 prodomain leads to tissue-specific loss of activity

Devorah C. Goldman¹, Renee Hackenmiller¹, Takuya Nakayama^1,*, Shailaja Sopory¹, Crispin Wong¹, Holger Kulessa^2, and Jan L. Christian^1,

¹ Department of Cell and Developmental Biology, Oregon Health and Sciences University, School of Medicine, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA.
² Vanderbilt University Medical Center, Division of Gastroenterology, D4108 Medical Center North, Nashville, TN 37232-2279, USA.

View larger version (59K): [in a new window]   Fig. 1. Generation of Bmp4S2G mice. (A) Schematic illustration of cleavage patterns of wild-type and S2G mutant forms of proBMP4. (B) Wild-type or mutant forms of the BMP4 precursor were expressed in HEK 293 cells and cleavage products analyzed by probing western blots of cell lysates with anti-HA antibody which recognizes an epitope tag present in the prodomain. Bands corresponding to uncleaved precursor, intact prodomain following cleavage at the S1 site, and intact prodomain following cleavage at the S2 site are indicated. Introduction of a point mutation [BMP4(mS2G)] that prevents cleavage at the S2 site still allows for cleavage at the S1 site whereas both sites are cleaved in native proBMP4. Mature BMP4 cleaved from either precursor migrates with an identical molecular mass (data not shown). (C) Genomic organization of the wild-type Bmp4 allele, the targeting vector and the Bmp4S2Gneo allele. The positions of the probes used for Southern analysis, and primers for PCR genotyping (arrows) are indicated. (D) Southern blot analysis of genomic DNA from targeted (H6, 2D8 and 2A10) or non-targeted (ES) ES cells. Genomic DNA was digested as indicated and probed with the internal (probe 2) or external (probe 3) genomic fragments shown in panel C. (E) PCR genotyping of progeny generated by interbreeding Bmp4S2G/+ mice. The PCR product generated by the mutant allele is 293 bp and that generated by the wild-type allele is 259 bp.

View larger version (54K): [in a new window]   Fig. 2. Reduced PGCs and phosphoSmad staining in Bmp4S2G/S2G embryos. (A) Graph of PGC number versus somite pairs for wild-type, Bmp4S2G/+ and Bmp4S2G/S2G embryos. (B) Immunostaining of E6 Bmp4+/+ and Bmp4S2G/S2G embryo sections with an antibody that recognizes the phosphorylated forms of SMAD1, 5 and 8. Staining in the proximal epiblast (arrows) and the visceral endoderm (arrowheads) is decreased in the Bmp4S2G/S2G embryo.

View larger version (101K): [in a new window]   Fig. 3. Testicular degeneration in Bmp4S2G/S2G adults. (A) Isolated testes from age matched wild-type and Bmp4S2G/S2G adult males. (B,C) Hematoxylin and Eosin-stained sections from age matched adult testis. (D-G) DAPI staining (D,F) and TUNEL staining (E,G) of sections from age matched adult testes. (H,I) Hematoxylin and Eosin-stained P6 testis sections from wild-type and mutant littermates. (J) Western blots of equivalent amounts of protein from wild-type and mutant P6 testes probed with an antibody directed against the mature domain of BMP4. The blot was reprobed for actin as a loading control.

View larger version (83K): [in a new window]   Fig. 4. Skeletal defects observed in Bmp4lacZ/+ mice are not present in Bmp4S2G/S2G mice. (A,B) Alcian Blue-stained E15.5 limbs. A postaxial cartilaginous element is indicated with an asterisk. (C-F) Dorsal views of skeletal preparations of cervical (C,D) and thoracic (E,F) vertebrae from adults. Yellow arrowheads mark vertebrae that have not fused and/or formed a dorsal spinous process. Black arrows indicate the 13th rib.

View larger version (62K): [in a new window]   Fig. 5. Defects in the allantois and placental vasculature of Bmp4lacZ/S2G embryos. (A,B) Expression of Gata4 in E8.75 embryos, analyzed by whole mount in situ hybridization. Arrows indicate the distal end of the allantois that has not undergone cavitation in Bmp4lacZ/S2G embryos. (C-H) ß-Galactosidase expression in whole-mount placentas isolated from Bmp4lacZ/+ or Bmp4lacZ/S2G embryos at E9.5 (C,D), E10.5 (E,F) or E11.5 (G,H). Inset in E and F shows a sagittal section through the placenta. (I,J) Whole-mount anti-PECAM staining of placentas from E11.5 littermates.

View larger version (94K): [in a new window]   Fig. 6. Defects in ventral body wall closure and eye development in Bmp4lacZ/S2G mutants. (A) E17.5 Bmp4lacZ/S2G embryo with complete failure of ventral body wall closure. (B) Coronal sections through E16 embryos. Arrowhead indicates umbilical artery, arrow denotes midgut, which has been enclosed by the ventral body wall in the Bmp4S2G/+ embryo but remains externalized in the Bmp4lacZ/S2G littermate. (C,D) E12.5 embryos showing loss of eye pigment in Bmp4lacZ/S2G embryos. (E,F) Expression of Tbx5 in E10.5 embryos, analyzed by whole-mount in situ hybridization. e, eye; he, heart; lb, limb bud. Insets show staining of the eye at higher magnification.

View larger version (155K): [in a new window]   Fig. 7. Ventricular septal defects in Bmp4lacZ/+ and Bmp4lacZ/S2G mutants. Hematoxylin and Eosin-stained coronal sections of an E16.5 Bmp4S2G/S2G embryo (A,D,G), an E17.5 Bmp4lacZ/+ embryo (B,E,H) and an E16.5 Bmp4lacZ/S2G embryo (C,F,I). (A-C) Anterior sections at the level of the pulmonary trunk (PT) and aorta (Ao) show normal outflow tract septation. (D-F) Sections through the muscular (*) and membranous (Mb) portion of the ventricular septum. Arrows indicate an aberrant opening where the membranous portion of the septum has not formed. (G-I) Sections through the atrioventricular valves (arrowheads) and atrial septa (arrows) showing normal development of these structures. LA, left atrium; Lu, lung; LV, left ventricle; RA, right atrium RV, right ventricle.