ARTICLE |
Correspondence to: Andrew Jheon, CIHR Group in Matrix Dynamics, 234 FitzGerald Building, 150 College Street, Faculty of Dentistry, University of Toronto, Toronto, ONT, Canada M5S 3E2.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bone morphogenetic proteins (BMPs) are characterized by their ability to induce osteoblastic differentiation. However, the mechanism of osteo-induction by BMPs has yet to be determined. Using differential display we previously identified AJ18, a zinc finger transcription factor, as an immediateearly response gene to BMP-7. AJ18 was shown to bind to the osteoblast-specific element2 (OSE2) and to modulate transactivation by Runx2, a master gene in osteoblastic differentiation. Here we describe the temporal and spatial expression of AJ18 in developing mouse tissues. AJ18 mRNA expression was observed in most tissues, except liver, and was generally highest early in embryonic development, decreasing markedly after parturition. Consistent with immunohistochemical analysis, AJ18 mRNA expression was highest in the brain, kidney, and bone of 17 dpc (days post coitum) embryos. In endochondral bones of embryonic and 4-week-old mice, immunostaining for AJ18 was strong in the nuclei of proliferating and pre-hypertrophic chondrocytes, and osteoblasts, whereas there was low or no staining in hypertrophic chondrocytes. In teeth of embryonic and 4-week-old mice, nuclear staining was observed in precursor and mature ameloblasts, odontoblasts, and cementoblasts, respectively. In addition, in 4-week-old mice staining of AJ18 was observed within alveolar bone cells and periodontal ligament cells. In general, the spatial expression of AJ18 in skeletal and non-skeletal tissues of mouse embryos showed striking similarity to the expression of BMP-7 mRNA. Therefore, the expression of AJ18 is consistent with its perceived role as a transcriptional factor that regulates developmental processes downstream of BMP-7. (J Histochem Cytochem 50:973982, 2002)
Key Words: AJ18, BMP-7, Runx2, transcription, repressor, bone, teeth, differentiation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ORGANOGENESIS involves temporospatially regulated morphogenesis of cells that differentiate to form organ-specific structures in the appropriate location (reviewed by
AJ18, a member of the growing family of KRAB/C2H2 zinc finger genes, was originally identified in fetal rat calvarial cells (FRCCs) as a target gene for BMP-7 (
To determine the physiological significance of AJ18 in bone development, preliminary analyses of AJ18 mRNA expression were performed in rat tissues (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse Genomic Library Screen
To characterize the mouse AJ18 gene, a mouse genomic library was prepared from 129SvJ mouse spleen DNA assembled into -phage FIX II vector (Stratagene; La Jolla, CA) and screened by plaque hybridization (
Mouse Tissue Preparation
Tissues from brain, heart, lung, skeletal muscle, cartilage, liver, thymus, kidney, calvaria, and long bones were dissected from mice at embryonic stages 15 and 17 dpc and at Days 2, 7, 15, and 23 after birth. Mouse embryos at 16 dpc and mandible and tibia from a 4-week-old mouse were isolated and fixed in 4% paraformaldehydePBS at 4C. The mandible and tibiae were demineralized in 12.5% (w/v) EDTA (pH 7.4), with the solution changed other day for 23 weeks. The embryos and tissues were embedded in paraffin. Serial 12-µm-thick sections were mounted on Superfrost/Plus glass slides (Fisher Scientific; Nepean, Canada) and stored at 4C until use.
RNA Extraction
Total RNA was isolated from mouse tissues using the thiocyanatephenolchloroform extraction method as described by
Northern Blot Hybridization
Northern blot hybridization was performed as described previously (-clone using primers 5'-GGCACA-CCTTTCATCTACGGCTCATCC-3' and 5'-ACGCCTGTATCCATCCCCACTGTTAAG-3', and was used as the template for probe synthesis. The intensity of the AJ18 mRNA bands was visualized with a PhosphorImager (Molecular Dynamics; Sunnyvale, CA) and quantified using the ImageQuant software (Molecular Dynamics). Relative expression levels of AJ18 mRNA were determined by normalization to expression levels of glyceraldehydephosphate dehydrogenase (GAPDH).
Anti-AJ18 Polyclonal Antibodies
Polyclonal antibodies were raised against peptides specific for rat AJ18 and affinity-purified as described in
IHC Analysis
Immunoperoxidase staining for AJ18 protein in tissue sections from 11, 12, 13, 14, 15, and 16 dpc mouse embryos and from 4-week mouse mandible and tibia was performed using the Vectastain ABC kit (Vector Laboratories; Burlingame, CA) according to the manufacturer's instructions. Tissue sections, prepared as described above, were rehydrated through incubation in graded ethanol to water and incubated in blocking solution (5% BSA, 2% normal goat serum) for 1 hr. Affinity-purified anti-AJ18-1 or anti-AJ18-2 antibodies were applied and tissue sections were incubated for 1 hr. The sections were washed and treated with biotinylated anti-rabbit IgG for 30 min, followed by incubation with peroxidase-labeled streptavidin for 30 min, and subsequently incubated with diaminobenzidine tetrahydrochloride (DAB) and H2O2 for 15 min. All incubations were performed at 21C. Various sections were counterstained with hematoxylin. All sections were visualized under a light microscope (Eclipse 400; Nikon Canada, Mississauga, Canada) and photographed using a Coolpix 950 digital camera (Nikon Canada). All immunostaining was performed at least three times on sections obtained from at least three individual mice (embryos and 4-week-old).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of Mouse AJ18
The sequence of the mouse AJ18 open reading frame (ORF) was deduced from an 10-kb
-clone that was isolated from mouse genomic screens. Because of the high conservation of the nucleotide sequence between mouse and rat AJ18, the mouse AJ18 gene was readily identified and the sequence of the ORF determined from a comparison of the mouse genomic sequence to the rat cDNA. This was simplified, in part, because the 11 C2H2 zinc finger motifs are over 97% identical and are encoded by a single exon. In total, the translated protein sequences of rat and mouse AJ18 share 91% identity and 94% similarity (Fig 1). Whereas the N-terminus, KRAB domain, and zinc finger regions of mouse and rat AJ18 are almost perfectly conserved, showing between 97% to 100% identity, the linker sequence between the KRAB domain and zinc finger motifs and the C-terminus show relatively low conservation with 72% and 79% identity, respectively.
|
Northern Blot Hybridization
In previous studies, expression of AJ18 mRNA in whole mouse embryos was first detected in 11 dpc embryos, reached a peak at 15 dpc, and decreased in 17 dpc embryos (
|
IHC Staining
In concert with Northern blot analyses, IHC analyses were performed on tissue sections of whole mouse embryos at various developmental stages and in neonatal tissues. Because the mouse and rat AJ18 sequences are highly conserved, the anti-rat AJ18 polyclonal antibodies raised to synthetic peptides 1 (residues 213) and 2 (residues 158169) should recognize both rat and mouse AJ18 protein. Affinity-purified anti-AJ18-1 and anti-AJ18-2 antibodies recognized mouse AJ18 protein (data not shown), although anti-AJ18-1 showed higher immunoreactivity, consistent with the 100% conservation of sequence between residues 2 and 13 (Fig 1). Therefore, all the tissue sections presented hereafter were immunostained using affinity-purified anti-AJ18-1 antibody. Strongest staining was obtained for all tissues at Day 16 dpc, at which time skeletal tissues could be analyzed at different developmental stages in the ribs. Consequently, the results present here are primarily for tissues at this stage (Fig 3 Fig 4 Fig 5, Fig 7, Fig 8, and Fig 10). Although staining for AJ18 was observed in the same tissues beginning at 1314 dpc mouse embryos, staining intensity was lower relative to 16 dpc mouse embryos, and the differential expression within and between tissues was less clear. To analyze expression of AJ18 in further developed tibial bone and teeth, sections from a 4-week-old mouse were immunostained (Fig 6 and Fig 9).
|
|
Expression of AJ18 Protein in Cartilage and Bone
Immunostaining of AJ18 was high in skeletal tissues of 16 dpc mouse embryos, as shown for bone of the maxilla and mandible, Meckel's cartilage, and ribs (Fig 3 Fig 4 Fig 5). During embryonic development, mesenchymal cells in the facial region condense to form nodules of differentiated osteoblasts that form the maxillar and mandibular bones of the upper and lower jaws, respectively. Expression of AJ18 was evident in the nuclei of mesenchymal cells adjacent to the bone surfaces, with stronger staining of the osteoblasts lining mineralized bone surfaces (Fig 3A and Fig 3C). AJ18 was also detected within some newly formed osteocytes, but no staining was apparent in the endothelial cells surrounding blood vessels or in loose connective tissue (Fig 3C). AJ18 was detected within the nuclei of the small chondrocytes of Meckel's cartilage, which forms the template for mandibular bone (Fig 3E). In identically prepared control sections incubated without the primary antibody, no signal was observed (Fig 3B, Fig 3D, and Fig 3F). Because the individual ribs form at different times, a progression of endochondral bone development could be seen at a single time point in a transverse section through a series of ribs. Thus, staining for AJ18 in the 10th, 11th, and 12th ribs showed protein in nuclei of cartilage and bone cells (Fig 4A). In the 12th rib, nuclear staining of AJ18 was evident in pre-hypertrophic chondrocytes (Fig 4B). In the 11th rib, which is further developed, the cartilage cells had undergone hypertrophy and cell nuclei were stained sporadically for AJ18 (Fig 4C). At this stage, when the cartilage had started to mineralize and the osteogenic periosteal cells had condensed, AJ18 was present in the nuclei of cells on the surface of the mineralizing bone. In the more advanced 10th rib, endochondral bone had formed centrally, replacing the mineralized cartilage, and the formation of periosteal bone was well established (Fig 4D). Staining for AJ18 was observed in cells present in bone marrow, in the periosteal cells on the surface of the rib bone, and in osteocytes present within periosteal bone. In these sections, comparable staining was also present in skeletal muscle tissue surrounding the ribs (Fig 4A) and was particularly strong in the outer epithelial layer of the embryo (Fig 4E). In skeletal muscle and especially in the epithelium, diffuse staining was seen in the cells, with nuclear staining in the epithelium being strongest in the basal cell layer.
A longitudinal section through the growth plate of the 3rd rib from a 16 dpc mouse embryo showed the developmental progression of cartilage cells through the formation of columnar proliferating chondrocytes to hypertrophic chondrocytes in the mineralized cartilage that is subsequently replaced by epiphyseal bone (Fig 5A). The strongest staining was seen in the proliferating chondrocytes with lower expression in hypertrophic chondrocytes (Fig 5A and Fig 5B), consistent with the staining in the 11th and 12th ribs. However, staining was high in the osteogenic cells in the primary spongiosa immediately below the calcified cartilage and in the subperiosteal bone collar formed by periosteal cells (Fig 5C). Staining in skeletal muscle was also observed.
To examine AJ18 expression at later stages of endochondral bone formation, sections of tibiae from a 4-week-old mouse were analyzed (Fig 6A). AJ18 staining was observed in some pre-hypertrophic and hypertrophic chondrocytes (Fig 6B), with strong staining visible in osteoblasts (Fig 6C). Staining for AJ18 was also present in the cartilage cells at the periarticular surface (Fig 6D). No staining for AJ18 was observed in osteocytes embedded within cortical bone (Fig 6E). These results were similar to those obtained in tibiae from a 4-week-old rat (
Expression of AJ18 Protein in Developing Teeth
Three principal stages (bud, cap, and bell) are recognized in the formation of teeth (reviewed by
Expression of AJ18 was also studied in the incisors and fully developed molars of a 4-week-old mouse (Fig 8A). In sections through the molar roots, staining for AJ18 was seen in the dental papilla, with strong staining in the odontoblasts lining the dentin. Strong staining was also apparent in the cells of the periodontal ligament and in osteogenic cells lining the surface of the alveolar bone. However, nuclear staining of osteocytes was weak (Fig 8B). In the continuously erupting incisor tooth, a continuum of cell differentiation associated with the formation of dentin and enamel was present only on the labial side of the mouse mandibular incisor. AJ18 staining was observed in the cells of the dental papilla, with strong staining in the newly differentiating odontoblasts (Fig 8C), which lose the staining after the formation of dentin has started. Anterior to this region, the nuclei of ameloblasts with their elongated cell bodies were strongly stained (Fig 8D and Fig 8E). Moreover, the staining intensity appeared to be retained as the ameloblasts progressed from the secretory stage, in which they appear highly ordered (Fig 8D), to the post-maturation stage, in which they undergo apoptosis and the columnar arrangement becomes disorganized (Fig 8E).
Expression in the Brain and Epithelium
Expression of AJ18 mRNA was highest in brain in the embryo and especially after birth, when expression in other tissues declined markedly (Fig 2). However, immunostaining sections of brain in 16 dpc embryos was variable, with the strongest staining in condensing cells that form the future cerebral cortex, midbrain, and tentorium cerebelli (Fig 9A and Fig 9C). In general, staining was not as strong as that seen in the osteogenic cells forming the calvarial bone and was much lower than the staining seen in the epithelium covering the cranium (Fig 9B). Because AJ18 mRNA expression appeared stronger in brain compared to bone, it is possible that protein translation in brain cells is less efficient than in bone cells, or that AJ18 may be highly expressed in specific regions of the brain that were not included in the sections analyzed. Relatively strong staining was observed in skin epithelia (Fig 9B). Although nuclear staining was present in the basal cells of epithelia, staining in the outer layers was diffuse, possibly due to cell death.
Expression in the Kidney, Eye, and Whisker Follicles
Northern blot analyses showed AJ18 mRNA expression in the kidney of mouse embryos at 15 and 17 dpc. Consistent with these results, relatively strong immunostaining for AJ18 was observed in cells within developing glomeruli of the kidney (Fig 10A). In the eye, specific staining was found in cells within the sclera, choroids, and vitreous body, with less staining in the retina (Fig 10C). AJ18 staining was also observed in the epithelial root sheath of developing whisker follicles (Fig 10E). The strong staining of nuclei in the whisker follicles is consistent with the general staining of epithelial cells, including the epidermal cells (Fig 3 and Fig 9), and of epithelial cells lining the intestine and lung alveoli (results not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies have characterized AJ18 as a zinc finger transcription factor that is upregulated in osteogenic cells induced to differentiate with BMP-7. Consistent with the expression of AJ18 mRNA during osteogenic differentiation by FRCCs in vitro (
Comparison of the protein sequences for AJ18 in rat and mouse revealed high conservation of amino acids (97100%) in the region of the C2H2 zinc fingers and the KRAB domain, signifying the functional importance of these regions in mediating DNA binding and transcriptional activity. In the linker region between the KRAB domain and the zinc fingers, and at the carboxy-terminal region, sequence conservation was much lower (7279%), indicating that the precise structure of these regions is of lesser functional importance. In previous studies (
Although BMPs are characterized by their bone-inductive activity (
From the IHC analysis of endochondral bone in 16 dpc mouse embryos, it is evident that AJ18 is initially expressed by proliferating chondrocytes and pre-hypertrophic chondrocytes, with sporadic expression in hypertrophic chondrocytes. The formation of cartilage in endochondral bones is controlled by a negative regulatory feedback loop involving PTH-related protein (PTHrP) and Indian hedgehog (Ihh) (
BMP-7 has also been shown to inhibit the terminal differentiation of chondrocytes in the periarticular region, independent of the Ihh/PTHrP/BMP-6 negative feedback loop via unknown inhibitory factors (
In summary, these studies show that the expression profile of AJ18 in embryonic tissues is consistent with the concept that AJ18 acts downstream of BMP-7 during tissue morphogenesis.
![]() |
Acknowledgments |
---|
Supported by a grant (MOP) from the Canadian Institutes of Health Research (CIHR). Andrew Jheon was the recipient of an Ontario Graduate Student Science and Technology (OGSST) scholarship.
Received for publication October 8, 2001; accepted February 6, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernier SM, Goltzman D (1993) Regulation of expression of the chondrocytic phenotype in a skeletal cell line (CFK2) in vitro. J Bone Miner Res 8:475-484[Medline]
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159[Medline]
Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747-754[Medline]
Dudley AT, Lyons KM, Robertson EJ (1995) A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev 9:2795-2807[Abstract]
Goldring MB (1999) The role of cytokines as inflammatory mediators in osteoarthritis: lessons from animal models. Connect Tissue Res 40:1-11[Medline]
Grimsrud CD, Romano PR, D'Souza M, Puzas JE, Reynolds PR, Rosier RN, O'Keefe RJ (1999) BMP6 is an autocrine stimulator of chondrocyte differentiation. J Bone Miner Res 4:475-482
Haaijman A, Karperien M, Lanske B, Hendriks J, Lowik CW, Bronckers AL, Burger EH (1999) Inhibition of terminal chondrocyte differentiation by bone morphogenetic protein 7 (OP-1) in vitro depends on the periarticular region but is independent of parathyroid hormone-related peptide. Bone 25:397-404[Medline]
Helder MN, Ozkaynak E, Sampath KT, Luyten FP, Latin V, Oppermann H, Vukicevic S (1995) Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development. J Histochem Cytochem 43:1035-1044
Hogan BL (1996) Bone morphogenetic proteins in development. Curr Opin Genet Dev 6:432-438[Medline]
Jheon A, Ganss B, Cheifetz S, Sodek J (2000) Identification of a novel zinc finger transcription factor target of BMP-7 in osteogenesis. In Goldberg M, Boskey A, Robinson C, eds. Chemistry and Biology of Mineralized Tissues. Rosemont, IL, American Academy of Orthopaedic Surgeons, 87-91
Jheon AH, Ganss B, Cheifetz S, Sodek J (2001) Characterization of a novel KRAB/C2H2 zinc finger transcription factor involved in bone development. J Biol Chem 276:18282-18289
Kim IS, Otto F, Zabel B, Mundlos S (1999) Regulation of chondrocyte differentiation by Cbfa1. Mech Dev 80:159-170[Medline]
Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M et al. (1996) PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273:663-666[Abstract]
Luo G, Hofman NC, Bronckers AL, Sohocki M, Bradley A, Karsenty G (1995) BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev 9:2808-2820[Abstract]
Peters H, Balling R (1999) Teeth: where and how to make them. Trends Genet 15:59-65[Medline]
Rodan GA, Harada S (1997) The missing bone. Cell 89:677-680[Medline]
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual. 2nd ed Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press
Takeda S, Bonnamy JP, Owen MJ, Ducy P, Karsenty G (2001) Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice. Genes Dev 15:467-481
Ten Cate AR (1994) Development of the tooth and its supporting tissues. In Ladig D, ed. Oral Histology. 4th ed St Louis, MO, MosbyYear Book, 58-80
Thomadakis G, Ramoshebi LN, Crooks J, Rueger DC, Ripamonti U (1999) Immunolocalization of bone morphogenetic protein-2 and -3 and osteogenic protein-1 during murine tooth root morphogenesis and in other craniofacial structures. Eur J Oral Sci 107:368-377[Medline]
Vainio S, Karavanova I, Jowett A, Thesleff I (1993) Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75:45-58[Medline]
Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ (1996) Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273:613-622[Abstract]
Watson RP, TekkiKessaris N, Boulter CA (2000) Characterization, chromosomal localization and expression of the mouse Kid3 gene. Biochim Biophys Acta 1490:153-158[Medline]