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First published online March 23, 2006
doi: 10.1242/10.1242/dev.02308

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Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis

¹ Department of Biomedical Engineering and Orthopaedic Research Center, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
² Laboratory of Connective Tissues Biology, Center of Biomedical Integrative Genoproteomics, University of Liège, 4000 Sart Tilman, Belgium.
³ Department of Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

View larger version (47K): [in a new window]   Fig. 1. Adamts2 and Adamts3 expression overlap with Col1a1 in the 7.5 dpc uterus. Note that Adamts3 expression is similar to that of Col1a1, whereas Adamts2 expression is different but has partial overlap. Asterisk indicates the embryo, which did not show expression. Scale bar: 100 µm.

View larger version (85K): [in a new window]   Fig. 2. Co-expression of Adamts3 and Col2a1 in musculoskeletal tissues. (A-G) In situ hybridization is used to show expression of Adamts3 in developing cartilage from 12.5 dpc to 17.5 dpc. In A-D, images include hybridization of Col2a1 to adjacent serial sections. Hoechst 33258 stained images are included on the far right for morphological correlation; the age of the embryo from which the section was taken is shown on the right. (A) Expression in initial sites of chondrogenesis in the developing ribs (arrows). (B) Expression in primordial cartilage of the ankle joint. T, tibia; Ts, Talus; C, calcaneus. (C) Expression in the proximal humerus growth plate. Note that expression is restricted to the proliferating zone (PZ). Hypertrophic zone chondrocytes (HZ) do not express Adamts3. Col2a1 is not expressed in the bone of the primary ossification center (B). Adamts3 is expressed in the perichondrium (arrow) and bone. (D) Proximal femur. The arrow indicates a tendon insertion site where Adamts3, but not Col2a1 is expressed. (E-G) 17.5 dpc skeletal elements, demonstrating that Adamts3 is not expressed by hypertrophic chondrocytes. (E) Absence of Adamts3 expression in the hypertrophic chondrocytes (HC) of the manubrium sterni. Note Adamts3 expression in developing intercostal muscle (arrow). (F) Ribs and vertebral lateral process. The rib elements contain hypertrophic chondrocytes (asterisk) that do not express Adamts3. By contrast, the lateral process of a thoracic vertebra (v) has small chondrocytes and these express Adamts3. The black oval indicates a spurious signal from erythrocytes. (G) Sagittal section through the basi-occiput (BO) and the dens (d) of the second cervical vertebra. Note the strong expression of Adamts3 in regions containing small chondrocytes and absence from the hypertrophic zone (asterisk). Expression is seen in the perichondrium (arrows). Scale bar: 100 µm.

View larger version (149K): [in a new window]   Fig. 3. Co-expression of Adamts3 and Col1a1 in the musculoskeletal system. All images are from 15.5 and 17.5 dpc embryos; Hoechst 33258-stained images are shown in the right-hand panel. (A) Expression in the appendicular skeleton (proximal humerus). Note that Adamts3 expression overlaps with Col1a1 in bone (B) and in the perichondrium around the hypertrophic zone and the periosteum (around bone). Chondrocytes in the proliferating zone (PZ) and hypertrophic zone (HZ) are indicated; arrows indicate mRNA in muscle. (B) Expression in the craniofacial skeleton. Note co-expression of Col1a1 and Adamts3 in bone (B) in the mandible and maxilla, as well as in developing masseter muscle (mt). (C) Expression in the proximal femur. The trochanteric fossa (TF), which contains the obturator tendons, is indicated by the arrow. The left panel in this figure is the same as that in Fig. 2D. (D) Expression in the developing Achilles tendon at 15.5 dpc. The image on the left shows Adamts3 expression in the ankle cartilage (C) and tendon (arrow); that on the right shows the Achilles tendon at a higher magnification (boxed area in middle panel). Scale bar: 100 µm.

View larger version (65K): [in a new window]   Fig. 4. Adamts3, not Adamts2, has a role in procollagen I processing in the skeleton. (A) Normal craniofacial development, bone structure and minimal tooth involvement in Adamts2-/- mice. mCT analysis and three-dimensional reconstruction of skulls of Adamts2-/- mice and wild-type (+/+) littermates do not show differences in overall patterning, size, dimensions or organization of any craniofacial elements. The lower panels show tooth morphology from littermates. Note the poorly defined topography of molars from Adamts2-/- mice, although the overall morphology is similar. (B) Adamts3, but not Adamts2 or Adamts14, was strongly expressed during tooth development in the dental papilla (DP), and was expressed less strongly in the ameloblasts (arrow). (C) Expression of ADAMTS3 in stably transfected 293 cells. Conditioned medium from cells was analyzed by western blotting with a polyclonal antibody to ADAMTS3. Note that upon treatment with PNGase F, ADAMTS3 migrates faster. Untransfected cells showed no protein. Molecular mass markers are indicated on the right. (D) Rescue of the procollagen I processing defect by ADAMTS2 and ADAMTS3 in dermatosparactic calf skin fibroblasts (DS). DS were plated alone or mixed with an identical number of HEK-293 cells stably transfected with an empty expression vector (Ct), or with the same vector encoding ADAMTS2 (TS2) or ADAMTS3 (TS3). After two days in culture, western blotting of a non-reducing SDS-PAGE gel was used to detect type I collagen. Note that the antibody used detects both 1 and 2 chains, but reacts more strongly with the 1 chain. The identity of collagen polypeptides associated with the cell layer or recovered from the conditioned culture medium is indicated between the two panels. (E) Rescue of the procollagen III processing defect by ADAMTS2 and ADAMTS3 in DS. Samples used in D were also analyzed by western blotting using an anti-collagen III antiserum. The lower amount of collagen synthesized by DS cultured alone, as compared to co-cultures, was repeatedly observed and may be related to the lower level of confluence in the monoculture. Alternatively, HEK-293 cells may secrete factors activating the synthesis of procollagen. Scale bar: 100 µm.

View larger version (91K): [in a new window]   Fig. 5. Expression of Adamts3 outside the musculoskeletal system. Hoechst 33258-stained images are shown in the right-hand panel. (A) Co-expression of Adamts3 and Col2a1 in the floor of the fourth ventricle in the hindbrain (arrow). (B) Expression of Adamts3 in the cerebral cortex. (C) Adamts3 expression in the ependyma of the subcommissural organ. III, third ventricle. (D) Adamts3 in dorsal root ganglia (drg). Note the sparse expression in the vertebral cartilage (v) that comprises mainly hypertrophic chondrocytes. (E) Expression of Adamts3 in the inner musculature of the urinary bladder. Note, in B-E, Adamts3 was not co-expressed with Col2a1. Scale bar: 100 µm.

View larger version (63K): [in a new window]   Fig. 6. Co-expression of Adamts2 and Col3a1, and deficiency of procollagen III processing in Adamts2-/- mice. (A,B) Adamts2 is co-expressed with Col3a1 in the arterial wall, as shown at two developmental stages: 12.5 dpc carotid artery (A) and 17.5 dpc pulmonary artery (B). L, lung. (C) Expression of Col3a1 and Adamts2 in lung. Note the expression in lung mesnchyme but not in the epithelium of bronchial tubes (asterisk). (D) Co-expression of Adamts2 and Col3a1 in the mesenchyme of the palate (P). Adamts2 is also expressed in the perichondrium of the basisphenoid (BS), at which location Col3a1 is absent. (E) Co-expression of Adamts2 and Col3a1 in the peritoneum lining the liver (L, arrow), and mesentery of the gut (G) and pancreas (Pa). (F) Co-expression of Adamts2 and Col3a1 within the subepithelial layer of the developing urinary bladder. Scale bar: 100 µm. In D-F, Adamts2 expression is weaker than in lung and arteries, and Col3a1 expression is also significantly weaker and more dispersed. (G) Defective procollagen III processing in tissues from Adamts2-/- mice. Western blot analysis of procollagen isolated from mouse tissues with 0.15 M NaCl is shown. The tissue origin of the sample and the respective genotype is shown above each lane; procollagen III and collagen III molecular species are indicated on the left. Scanning densitometry of the different collagen III signals was used to calculate the proportion of fully processed molecules in the various tissues (lower panel). (H) Defective procollagen III processing in newly synthesized collagen in Adamts2-/- mice. Protein was extracted from sponge granulomas and western blotting used to define the molecular species of procollagen III that were present. Sponge granulomas from two Adamts2-/- mice and their wild-type littermates are shown. Scale bar: 100 µm.

View larger version (73K): [in a new window]   Fig. 7. Histology and ultrastructure of lung and aorta in Adamts2-/- mice and wild-type (+/+) littermates. (A) Comparative lung histology from Adamts2-/- and their wild-type (+/+) littermates. The distal air spaces are much larger in the mutant mice, but there is no inflammatory exudate. (B) Ultrastructure of an alveolar wall, demonstrating comparable basement membranes (arrows) in Adamts2-/- and wild-type mice. E, erythrocyte. (C) Methylene blue-azure B stain for elastin in Adamts2-/- and wild-type aortas demonstrates comparable aortic wall thickness and elastin lamellae (arrow). (D) Electron micrograph of collagen fibrils in cross section (in area marked by the asterisk) demonstrates comparable size and shape, and uniformity of fibril diameter. Scale bar: 100 µm in A,C; 500 nm in B,D. (E) Western blot analysis of procollagen III (above) and I (below) processing in mouse lungs. Samples from wild-type and mutant newborn and adult mice are illustrated. (F) Western blot analysis of procollagen III (above) and I (below) processing in the mouse aorta. The various molecular species of collagen are indicated.

View larger version (73K): [in a new window]   Fig. 8. Procollagen N-propeptidase expression in skin. D, dermis; the white dashed line indicates the line of demarcation of epidermis and dermis. (A) Co-expression of Col3a1 and Col1a1 in the dermis of the skin of a 17.5 dpc embryo. Adamts2, but not Adamts14 or Adamts3, mRNA was detected in the dermis. (B) Adamts2 and Adamts14 mRNA, but not Adamts3 mRNA, is detectable in the dermis of two-week-old skin. Scale bar: 100 µm.

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