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Expression pattern of Dlx3 during cell differentiation in mineralized tissues
Bone 37 (2005) 799 – 809 www.elsevier.com/locate/bone
Expression pattern of Dlx3 during cell differentiation in mineralized tissues Sonia Ghoul-Mazgar a,b , Dominique Hotton a , Frédéric Lézot a , Claudine Blin-Wakkach c , Audrey Asselin a , Jean-Michel Sautier a , Ariane Berdal a,⁎ a
Laboratoire de Biologie Oro-faciale et Pathologie INSERM U 714, 15-21 rue de l'Ecole de Médecine 75270, Universités Paris 7 and Paris 6-IFR58, Paris Cedex 06, France b Laboratoire d'Histologie-Embryologie, Faculté de Médecine Dentaire de Monastir, Tunisia, France c Génétique et pathologies moléculaires CNRS-FRE 2720 Faculté de Médecine de Nice, Université Sophia-Antipolis, Nice, France Received 9 September 2004; revised 21 March 2005; accepted 30 March 2005 Available online 19 September 2005
Abstract The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1—its role in skeleton patterning, 2—its expression in dental epithelium and mesenchyme during morphogenesis, 3—the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4—recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid–osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton. © 2005 Elsevier Inc. All rights reserved. Keywords: Dlx; Enamel; Dentin; Cartilage; Bone
Introduction The Dlx gene family, related to Distal-less homeobox gene in Drosophila, encodes for proteins that act as transcription activators [1,2]. In vertebrates, there are six Dlx genes arranged in three clusters (Dlx1/Dlx2, Dlx3/ Dlx4 and Dlx5/Dlx6) [3]. Paired genes are localized on three distinct chromosomes with an inverted configuration and are separated by a short intergenic region [3,4]. ⁎ Corresponding author. Fax: +33 1 44 07 14 21. E-mail address: [email protected] (A. Berdal). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.03.020
Sharing cis-regulatory elements between members of Dlx bigene cluster may contribute to an observed overlap in gene expression pattern and a partial functional redundancy [5]. Dlx genes are key-players in development [6–10]. They are expressed in overlapping but distinct domains, primarily in the forebrain, branchial arches, sensory organs, limbs and tissues derived from epithelial–mesenchymal interactions [9,11]. In particular, Dlx genes are involved in early vertebrate morphogenesis of the head and dentition [12– 14]. Dlx homeobox genes have also been shown to be expressed during the morphogenesis of the distal regions of
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extending appendages which involves bone anlage formation [2,15]. The contribution of Dlx genes to skeletal morphogenesis has been established by the phenotypes of their invalidation in transgenic mice. Double Dlx1/Dlx2 null mutants exhibit major and site-specific defects in teeth and other craniofacial skeletal units [8]. Double targeted inactivation of Dlx5 and Dlx6 genes results in severe craniofacial, axial and appendicular skeletal abnormalities leading to perinatal lethality [15]. Non-redundant functions of Dlx homeoproteins have been documented in Dlx5−/− where a delay in osteoblast differentiation is observed [16,17]. Dlx genes have also been postulated to regulate cell differentiation in the skeleton including bone [18], cartilage [19] and tooth [20,21]. Compared to developmental studies, relatively few studies have focused on Dlx genes and proteins in the later process of biomineralization either in vitro [18,22] or in vivo [23]. Considerable attention has been paid to Dlx2 and Dlx5 and, very recently, during revision of this article, to Dlx3 [24]. Dlx3, a member of the Dlx family, is a transcription factor described in the neural crest and implicated during development of epithelial tissues such as skin, hair follicles, otic and olfactory placodes, limb bud and tooth germ. In epidermis, Dlx3 homeoprotein triggers the terminal differentiation of keratinocytes [25]. Gene-targeted mice are available, but their skeleton cannot be studied as loss of Dlx3 function is associated with lethal development due to its role in the placenta [26]. However, disturbed expression of Dlx3 is associated with skeletal defects observed in several mutant mice, inactivated for other genes (foo, [27], Hand2, [28]). Notably, the endothelin-1 signaling pathway has been well characterized and modulates Dlx3 expression (sucker mutation, [29], edn1downstream targets, [28]; Galphaq- and Galpha11-deficient embryos, [30]). The presence of Dlx3 mRNA in tooth germ has been reported during the bud, cap and bell stages of development ([11,21], this study). In humans, disruption of the Dlx3 coding sequence is associated with an inherited autosomal dominant disorder, Tricho–Dento–Osseous syndrome (TDOS). Despite the fact that the observed defects involve not only ectodermal derivatives, but also bone and teeth [31,32], little is known about this transcription factor in the normal skeleton and more specifically during cell differentiation and mineralization of extracellular matrices. TDOS is associated with defects in the craniofacial skeleton, which is essentially formed by a membranous bone formation process [32]. Furthermore, the detection of altered axial skeleton osteogenesis and the observed high mineral density in axial bone of known TDO individuals have recently suggested a role of Dlx3 in endochondral ossification [33]. Based on (1) the importance of Dlx3 during skeletal patterning [34], (2) the phenotype of TDOS related to Dlx3 mutations and (3) the emerging role of Dlx
homeoproteins in bone formation [35], the present study was designed to explore Dlx3 expression patterns in mineralized tissues in vitro and in vivo. The diverse secretory cells of mineralized tissues were shown to systematically express Dlx3. This investigation was made possible by the availability of differentiated cells from several sources according to the tissue-type: sampling of dental cells by microdissection of murine mandibular incisors, a classical experimental model system used to study the regulation of gene expression in ameloblasts and odontoblasts [36] and primary culture of craniofacial skeletogenic cells: calvarium osteoblasts for membranous bone formation [37] and nasal septum chondrocytes for endochondral bone formation [38]. During revision of this article, an in vitro study reported the direct role of Dlx3 in bone formation and upregulation of bone-related genes during osteoblast differentiation, via either protein–DNA or protein–protein interactions [24]. Our data show that, in vivo, the temporal window of Dlx3 homeoprotein expression varies specifically according to the terminal differentiation for each cell-type studied: ameloblasts, odontoblasts, osteoblasts and chondrocytes.
Materials and methods Preparation and characterization of the human Dlx3 probe Human samples A limited number of antenatal craniofacial tissues, excluding brain, were collected at Robert Debré hospital according to French ethical rules, and were used for total RNA extraction and generation of a Dlx3 riboprobe. Some samples from 9-week-old human embryos were also used for in situ hybridization [39,40]. Construction of the Dlx3 probe Two μg of total RNAs prepared using TRI-REAGENT protocol (Euromedex, Strasbourg, France) were reversetranscribed with an oligo(dT) primer according to the manufacturer's instructions (Invitrogen, San Diego, CA). Double-strand cDNA was amplified by a PCR with a regime of 94°C, 30 s (denaturing), 55°C, 30 s (annealing) and 72°C, 7 min (extension) for 35 cycles using sense P1dlx3 5′AAGGTCCGAAAGCCGCGTA-3′ and antisense P2dlx3 5′-CTGCTGCTGTAAGTGGGGT-3 (Genbank access number: NM-005220). The expected size (414 bp) for amplified PCR fragments was controlled on 2% agarose gel. The larger expected size (1947 bp including intron 2) of Dlx3 genomic DNA allowed verification of the absence of DNA contaminations in RNA extracts. Control PCR was also performed without reverse transcription, in order to exclude DNA contamination in RNA extracts. The amplified fragments were isolated and subcloned into pCR 2.1 plasmid (Invitrogen). In-frame cloning was confirmed by sequencing (Genome express, France).
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Northern blotting Ten μg of total RNA isolated from a 9-week-old embryo was fractionated electrophoretically into a 1% agarose formaldehyde gel and transferred onto nylon membranes (Amersham, Les Ulis, France). Membranes were successively prehybridized and then hybridized with antisense Dlx3 probes 32P-labeled by random priming (RediprimeII, Amersham Pharmacia Biotech, France) synthesized from pCR 2.1 plasmid after linearization with BamH1 restriction endonuclease and using T7 RNA polymerase (Gibco BRL, Life Technologies, Cergy-Pontoise, France). The hybridized blots were autoradiographed using Kodak films (Amersham).
Preparation of dental and chondrogenic cells for RT-PCR analysis
Investigation of in vivo Dlx3 expression
Cell culture method Chondrocytes were enzymatically isolated from the nasal septum of 21-day-old fetal Sprague–Dawley rats, as
Animals C57 Bl6 mice (Charles River Breeding Laboratories, France) were studied from Theiler developmental stage E14.5 to 1 day post-natally. Embryos were fixed with 4% paraformaldehyde immersion and cryostat sectioned [39,40]. Some embryos of the same developmental stage were also paraffin-embedded for immunohistochemistry [41]. In situ hybridization The Dlx3 antisense RNA digoxigenin-labeled probe (414 bp) was synthesized as described for the Northern blotting but using digoxigenin-UTP (Boehringer-Mannheim, Meylan, France). In situ hybridization was performed as previously described [39,40]: cryostat sections were hybridized with 30 μl of digoxigeninlabeled probes diluted to 1:200, and the reaction was revealed by anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche Diagnostics, Meylan, France). Sections were dehydrated, mounted under a coverslip and photographed. Immunohistochemistry Anti-Dlx3 polyclonal antiserum raised against a 16amino-acid synthetic peptide (aminoacids 242 to 256) of the murine Dlx3 protein (kindly provided by Maria Morasso, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland, USA; described in [41]) was used. Sections were deparaffinized, rehydrated and treated with citrate buffer (10 mM, pH 6) in the Microwave [42]. Sections were washed with 0.1 M phosphate-buffered saline-pH 7.3 and treated for 5 min with H2O2 in methanol then incubated for 1 h with the primary antibody and, after rinsing, were incubated for 10 min with streptavidin–peroxidase conjugated secondary antibodies (Amersham, France). The immune complexes were detected by a diaminobenzidine staining kit (DAKO Corporation, Carpinteria, CA, USA). Non-relevant rabbit immunoglobulins were used as negative control.
Microdissections Mandibles and alveolar bone of 56-day-old male C57Bl6 mice were removed under a dissecting microscope. The incisor was treated as follows: an apical portion containing the odontogenic organ and early mineralization zone of enamel and dentin was excluded in order to avoid epithelial– mesenchymal contamination. Lateral cuts were made along the cemento-enamel junction and the enamel organ was scraped from the labial part of the incisor. Finally, after crack opening the dentin, the dental mesenchyme was collected.
Table 1 Primers for RT-PCR assays Gene
Species
Size (bp)
Oligonucleotides
Dlx3
Rat
404
Mouse
414
Dlx2
Mouse
430
OC
Mouse
257
Amg
Mouse
370
ALP
Mouse
525
Calbindin D-28k
Mouse
480
GAPDH
Mouse
824
Rat
401
Rat
213
Sense primer: 5′-AAGTTCGTAAACCCAGGAC-3′ Antisense primer GGTGGGAATTGATTGAGCT Sense primer: 5′-AAGGTCCGAAAGCCGCGTA-3′ Antisense primer: 5′-CTGCTGCTGTAAGTGGGGT-3′ Sense primer: 5′-TCCTACCAGTACCAAGCCA-3′ Antisense primer: 5′-AAGCACAAGGTGGAGAAGC-3′ Sense primer: 5′-CTCACTCTGCTGGCCCTG-3′ Antisense primer: 5′-CCGTAGATGCGTTTGTAGGC-3′ Sense primer: 5′-GGATCAAGCATCCCTGAGCTTCAG-3′ Antisense primer: 5′-ATCATTGGTTGCTGGGGGCGCAC-3′ Sense primer: 5′-CATCTGGAACCGCACGGAAC-3′ Antisense primer: 5′-GCCTGGTAGTTGTTGTGAGC-3′ Sense primer: 5′-ACGGAAGTGGTTACCTGGAA-3′ Antisense primer: 5′-CACACATTTTGATTCCCTGG-3′ Sense primer: 5′-TTCCAGTATGATTCCACTCA-3′ Antisense primer: 5′-CTGTAGCCATATTCATTGTC-3′ Sense primer: 5′-GACCCCTTCATTGACCTCAACTA-3′ Antisense primer: 5′-AAGTTGTCATGGATGACCTTGGC-3′ Sense primer: 5′-GTCTCTTGCTAGAAGAGA-3′ Antisense primer: 5′-GCCTGCAGGGAAGGTCAT-3′
Ihh
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previously described [38,43,44]. Briefly, nasal septum was aseptically dissected and fragments were incubated in phosphate buffered solution with collagenase (400 U/ml) and hyaluronidase (750 U/ml) for 2 h at 37°C. Cells were then dissociated from the cartilage fragments and plated at 3.5 × 104 cell/cm2. The culture medium used was Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen), 10 mM β-glycerophosphate (Sigma), 50 μg/ml ascorbic acid (Sigma) and 50 U/ml of penicillin-streptomycin (Invitrogen). Cells were cultured for up to 21 days in a humidified atmosphere of 5% CO2 in air at 37°C. Culture media were changed 24 h after seeding and at 48-h intervals thereafter. This model has been established to represent the process of endochondral bone formation [38,43,44]. RT-PCR and real-time PCR analysis of murine tissues and cells As described for human cells (see previous chapter on construction of the Dlx3 probe), total RNAs were extracted,
reverse -transcribed and PCR was performed for 30 cycles. Table 1 summarizes the primers used, the sizes of the expected amplicons and the temperatures used. Real-time PCR was performed for Dlx3 and Indian Hedgehog (Ihh), using a Light cycler™ (Roche Diagnostic). Real-time RTPCR using SYBR Green I reaction was performed by a standard protocol. 2.5 μl of cDNA diluted at 1/100 was added to a 7.5-μl reaction mixture including MgCl2 at the optimal concentration (3–5 mM), each primer at the optimal concentration (4, 6 or 8 μm) and 1 μl of a Light cycler-DNA Master SYBR Green I (Roche Diagnostics) and incubated in a Light cycler ™ under the following conditions: at 95°C for 10 min as the denaturation program, followed by 40 or 50 cycles of 95°C for 10 s, each at the optimal annealing temperature for 10 s, 72°C for 10 s. The optimal conditions for each gene amplification and quantification are summarized for underlined primers in Table 1. The fluorescence measurement was recorded at the end of each cycle.
Fig. 1. Construction and validation of the Dlx3 probe. DLX3 probe was synthesized from a 9-week-old human embryo. The double-strand cDNA obtained was detected on 2% agarose gel, at the expected size of 414 bp (A). The absence of contamination with genomic DNA was confirmed by the absence of a 1947-bp amplicon. PCR performed without previous reverse transcription also demonstrates the absence of PCR amplification products (data not shown). Total cellular RNA (10 μg) from a 9-week-old human embryo was loaded onto a 1% formaldehyde agarose gel and hybridized with Dlx3 probe for Northern analysis (B). In situ hybridization using RNA digoxigenin-labeled probes on 9-week-old human embryo serial cryostat sections showed Dlx3-positive structures identified during bud (C) and cup stages (D) of dental development and in the palatal bone process (E).
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Results Generation and validation of the Dlx3 probe during human antenatal development (Fig. 1) 414 bp PCR amplicons were obtained from human samples (Fig. 1A), and used as probes by cloning in pCR2.1 plasmid. Antisense riboprobes allowed visualization of the Dlx3 transcript (≈4500 bp) in human orofacial samples from 9-week-old embryos (Fig. 1B). In situ hybridization was also performed with the same riboprobe in human samples (Figs. 1C, D and E). Epithelial cells of tooth bud (Fig. 1C) and cap stages (Fig. 1D) contained Dlx3 RNAs. Ossification centers in the maxilla were also Dlx3-positive (Fig. 1E). Distribution of Dlx3 mRNA and protein during tooth morphogenesis and cell differentiation in mice (Fig. 2) In view of the ethical limitations on human studies, Dlx3 expression was more systematically analyzed in mice. Data on epidermis (non shown) were identical to the results of other published studies [41]. As previously shown by in situ hybridization [21], Dlx3 expression was consistently observed at the mRNA (Fig. 2A) and protein levels (Fig. 2B) on serial sections of first molar tooth germs at Theiler stage embryonic day 15.5 (E15.5) in mice. The relative expression levels of Dlx3 transcripts were still high
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at the later bell stage of the first molar of 1-day-old mice (Fig. 2C), but were different in dental epithelium and mesenchyme (Fig. 2D). Dlx3 transcripts appeared to be regularly expressed in differentiating and differentiated ameloblasts (Fig. 2C), but were down-regulated during odontoblast polarization (Fig. 2D). Dlx3 expression was also analyzed by RT-PCR in microdissected murine dental cells (Fig. 2E). Mouse incisors were used for this study, as they are an established model for RNA investigation. The entire developmental sequence of amelogenesis and dentinogenesis is recapitulated in this experimental model system and distributed along the labial aspect of the mandibular incisor. The size of these continuously erupting teeth allows microscopic dissection of epithelial (forming enamel) and mesenchymal (forming dentin) cells. The amplicons shown in figure F illustrate the absence of epithelial–mesenchymal cross-contaminations during microdissection procedures, as established by several studies. Some of these genes are co-expressed in mesenchyme and epithelium (calbindinD-28k, alkaline phosphatase, and amelogenin), as established in previous reports [36,40, 45]). In contrast, Dlx2 homeogene, which is exclusively present in the epithelial compartment [23], was only detected in microdissected epithelium. Conversely, osteocalcin which is exclusively synthesized by odontoblasts, showed amplified cDNA exclusively in the corresponding mesenchyme. This method of validation confirmed co-
Fig. 2. In vivo investigation of Dlx3 transcript and protein in dental tissues. On serial sections of first molar tooth germs at Theiler stage 15.5, Dlx3 transcripts (A) and protein (B) were both detected in dental epithelium and mesenchyme. This expression persisted until the later bell stage observed 1 day post-natally (C). The highest magnification of the box in C shows that, in preodontoblasts (pod) and in undifferentiated ameloblasts (Am), transcripts were strongly expressed (D). However, in differentiated and polarized odontoblasts (od), this expression was absent. Phenotypic transcripts expressed either in mice microdissected dental mesenchyme (M) or epithelium (Ep) were analyzed by RT-PCR (F). Agarose gel analysis showed that Dlx3, ALP, Amg and calbindin 28-k were detected in both tissues, whereas Dlx2 was only detected in dental epithelium and OC, only in dental mesenchyme. GAPDH levels served as a loading control.
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expression of Dlx3 in both the mesenchymal and epithelial compartments.
Dlx3 protein expression during endochondral ossification (Fig. 4)
Dlx3 protein expression during intramembranous bone formation (Fig. 3)
Dlx3 protein expression was observed in the five classically described zones in the growth plate cartilage, as exemplified in murine ribs at Theiler stage E15.5 (Fig. 4F). Dlx3 protein was not detected in the resting zone (Fig. 4A). In the proliferative zone, Dlx3 protein was detected (Fig. 4B) and strongly expressed in the pre-hypertrophic cartilage zone where chondrocytes became much larger (Fig. 4C). No Dlx3 protein was detected in the calcified cartilage zone (Fig. 4D). Bone collar produced in the deep portions of the perichondrium surrounding the cartilage, was also analyzed. The newly differentiated periosteum covering newly deposited bone formed a continuous layer of Dlx3-positive osteoblasts over bone collar (Fig. 4D). A comparative study of various long bones in the autopodium and ribs demonstrated two distinct steps in growth plate cartilages, as shown in Figs. 4G to J. Figs. 4G and H illustrate the absence of Dlx3 labeling in the perichondrium, i.e., in the future bone collar and periosteum,
Frontal sections of the palate visualized Dlx3 labeling at the site of initiation of membranous ossification (Fig. 3A). Serial sections detected Dlx3 protein at sites of mesenchymal condensation (Fig. 3B) and in bone trabeculae containing positive cells (Fig. 3C). At the same stage of development, in situ hybridization showed a similar distribution for Dlx3 transcripts in relation to cell differentiation from E14.5 to 1day-post-natally (Fig. 3D). In forming alveolar bone, Dlx3 transcripts were clearly localized in cells lining bone trabeculae (Fig. 3E). Differentiating (Fig. 3F) and differentiated osteoblasts (Fig. 3G) also appeared to express Dlx3 homeoprotein. Finally, the recently embedded osteocyte, also called osteoid–osteocyte, contained Dlx3 RNAs, while more mature osteocytes were negative in mice and even in human samples (Fig. 3H).
Fig. 3. Immunohistochemical detection of Dlx3 protein and in situ hybridization of Dlx3 at Theiler stage 15.5 in mouse intramembranous bone. During palatal (p) membranous ossification process (A), serial frontal sections of Theiler stage E15.5 mouse embryos showed that Dlx3 protein was detected in the cell condensation (B) and the osteoblast lining bone trabeculae (t) of the maxilla (C). Transcripts were also detected in the osteoblast lining trabeculae (t) (D). Later, in 1-day-old mice, Dlx3 transcript expression was also detected in osteoblasts lining alveolar bone trabeculae (t) (E). Osteoblast progenitors (obp) (F), osteoblasts (ob) and newly differentiated osteocytes (noc) appeared to express Dlx3 protein (vertical arrows), whereas well differentiated osteocytes (oc) (horizontal arrows) did not (G). During palatal membranous ossification of a 9-week-old human embryo, Dlx3 transcripts were also detected in osteoblast progenitors (obp), osteoblasts (ob) and newly differentiated osteocytes (noc), but were absent in well-differentiated osteocytes (oc) (H). M: oral mesenchyme; OE: oral epithelium.
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Fig. 4. Immunohistochemical detection of Dlx3 protein at Theiler stage 16.5 in endochondral bone ossification centers. Growth plate cartilage accompanying endochondral bone ossification was observed on ribs (A–F and I–J) and fingers (G–H). Dlx3 protein was absent from the resting zone (A ×1000), but was observed in the proliferative zone (B ×1000), strongly expressed in the pre-hypertrophic chondrocytes (C ×1000) and was not detectable in the hypertrophic and calcified cartilage zones (D ×1000). Bone collar presented a continuous layer of Dlx3 positive osteoblasts under the periosteum (E ×1000). Underneath the perichondrium that covers younger cartilage, only prehypertrophic cells expressed Dlx3 (G ×400 and H ×1000). Osteoblasts of newly-formed bone collar (I ×1000) and the central primary ossification point (J ×1000) also expressed Dlx3 protein.
before osteoblast differentiation. In contrast, Fig. 4I shows immunopositive osteoblasts of the newly formed bone collar and Fig. 4J shows similar appearance in the central primary ossification point.
was used for biomaterial investigation [44]. Ihh (Fig. 5E) showed a distinct upregulation with a maximal level at day 12. Interestingly, Dlx3 showed the same modulation pattern of expression levels as Ihh (Fig. 5E).
RT-PCR of Dlx3 and Ihh cDNA in nasal septum cartilage cells (Fig. 5)
Discussion
In primary cultures, well differentiated osteoblasts obtained from calvarium (data non shown) and rat nasal septum chondrocytes (Fig. 5) appeared to express Dlx3 transcripts. Rat nasal septum culture can be used to assess the chondrocyte life-cycle during the process of endochondral bone formation. Several stages are distinguished: early stages of mesenchymal precartilaginous condensation (Fig. 5A), differentiation of chondrocytes (Figs. 5B and C) and finally extracellular biomineralization (Fig. 5D). This system can also be used to characterize molecular events related to chondrocyte cell stages by real-time RT-PCR showing types II and X collagens, aggrecan, Runx2 and Ihh expression and
This descriptive study focused on the expression pattern of Dlx3 homeoprotein and transcript in Dlx3 target-tissues, as illustrated by the TDOS phenotype. These tissues include: (i) epidermis and hair follicle, which have been extensively characterized in other studies [41], (ii) dental epithelium and mesenchyme [11,21] and (iii) forming bone, which have been analyzed previously [34] albeit in less detail. The initial step of this study was therefore designed to verify the existence of Dlx3 transcripts in the 4 skeletogenic cell types studied by RT-PCR. Interestingly, during revision of this manuscript, publication highlighted Dlx3 expression and function in osteoblasts [24]. Driven overexpression and
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Fig. 5. Analysis of Dlx3 and Ihh expression in primary cultures of rat chondrocytes. (A) Day 5 in culture: precartilaginous condensations are visible in the confluent cell layer. Precartilaginous condensations are indicated with white arrows. (B) Day 8 in culture: phase bright colonies of differentiating chondrocytes are present in the culture dish. (C) Day 12 in culture: differentiating areas appear to be more numerous in the cell layer. (D) Day 21 in culture: the differentiating areas are enlarged. Dark areas (stars) are visible corresponding to mineralization of the matrix. (E) Gene expression level (relative to GAPDH) of Dlx3 and Ihh were determined at days 5, 8, 12 and 21, using real-time PCR. The signaling molecule Ihh was strongly expressed at day 12 and a similar expression pattern was observed for Dlx3 transcript.
knock-down of Dlx3 in vitro established that Dlx3 controls the appearance of osteogenic markers [24]. The present study allowed characterization of Dlx3 mRNA in other mineralized tissues using various experimental model systems, for enamel (microdissected dental epithelium, [36]), dentin (microdissected dental mesenchyme; [36]) and endochondral bone formation (primary culture of nasal septum chondrocytes; [38]). The cDNA and antibodies used in this study were validated by the identity of our results and available data for Dlx3 mRNA or protein in tooth [11,21], osteoblasts [24] and epidermis [41], (this study, data non shown) [6]. In situ hybridization and immunolabeling identified Dlx3 in ameloblasts, differentiating odontoblasts, osteoblasts and finally chondrocytes. These findings therefore provide evidence that Dlx3 is not only expressed during early skeletal patterning, but also in all mineralized tissues studied, inside secretory cells and at later and distinct differentiation stages. While some functional redundancy for various Dlx homeoproteins has been illustrated using exclusive [16] and combined null mutations during early patterning [8,15] distinct Dlx homeoproteins have been shown in vitro to be instrumental in osteoblast [24,46–49] and chondrocyte [35,50] physiology. Dlx2 and Dlx5 are involved in the osteoinductive cascade of BMP2 [47]. In osteoblasts, these two homeoproteins as well as Dlx3 play a role in the transcriptional activity of osteocalcin gene and mediation of BMP2-induced Runx2 expression [48,49]. BMP2 has also
been shown to significantly increase Dlx3 expression during induced osteoblast differentiation [24]. Dlx3 protein stimulates osteocalcin promoter activity and Dlx3–Runx2 interaction inhibits the Runx-mediated transcription of osteocalcin gene [24]. During endochondral bone formation [35], Dlx5 appears to control chondrocyte maturation. This function has also been proposed in the odontoblast lineage, where (i) Dlx5 downregulation occurs during odontoblast terminal differentiation [20], similar to the downregulation of Dlx3 in our study, and (ii) a matrix protein expressed in differentiated odontoblasts, osteocalcin, is negatively regulated by Dlx5 [51]. The present study provides a comparative overview of Dlx3 involvement in various mineralized tissues, which raises the question of their significance in each tissuespecific context. In tooth formation, while Dlx3 was continuously expressed in differentiating and differentiated ameloblasts, it appeared to be down-regulated during odontoblast polarization. Consistently with the present data, dental anomalies associated with 4 bp deletion of human Dlx3 gene [31,32] consist of small, yellow-brown teeth with thin hypoplastic enamel (i.e. affected enamel secretion) and large taurodontic pulp chamber (i.e. affected dental pulp cell differentiation). Dlx3 therefore appears to control distinct formation stages during the process of odontogenesis depending on the tissue. In secretion stage ameloblasts, the Dlx3 pattern and enamel phenotype of TDOS suggest that
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Dlx3 controls enamel thickness. Enamel thickness is dependent on the secretion of the major enamel protein: amelogenin [52]. The presumed gene positively regulated by Dlx3 would therefore be the amelogenin gene. In secretion stage ameloblasts, a combinatorial role for Dlx homeoproteins may also be proposed, as quantitative studies have previously established an inverse relationship between Dlx2 expression levels and enamel thickness [23]. This type of developmental stage specification for Dlx3 involvement in ameloblasts and odontoblasts raises the question of its role in osteoblasts and chondrocytes during the respective processes of membranous and endochondral bone formation, as recent clinical investigation of TDOS defects have established that not only craniofacial [32], but also appendicular [33] skeleton formation is affected by DLX3 gene mutations. The appendicular skeleton is formed by the endochondral bone formation process which involves chondrocytes. In the present study, in the osteoblastic lineages, in contrast with odontoblasts where Dlx3 protein was down-regulated in terminally differentiated cells, Dlx3 protein was expressed from the preosteoblast stage until the osteoid–osteocyte stage. Down-regulation of Dlx3 protein was observed only in mature osteocytes. The present in vivo study provides a detailed description of Dlx3 protein expression levels in the osteoblast–osteocyte maturation process, which is complementary to recently published functional studies on Dlx3 in osteoblasts [24]. In endochondral ossification, Dlx3 protein appeared to be upregulated in prehypertrophic chondrocytes and its expression levels decreased in hypertrophic chondrocytes, in vivo and in vitro. Indian hedgehog (Ihh) is one of the main regulators of long bone development and like Dlx3, it is expressed by chondrocytes leaving the proliferative pool (prehypertrophic chondrocytes) [53]. Ihh couples chondrogenesis to osteogenesis in endochondral bone development, perhaps in synergy with BMP2 [54] and potentially Dlx3, as suggested here. Indeed, PCR data showed a parallel variation of Dlx3 and Ihh transcript levels which were maximal at day 12 in rat chondrocytes in vitro. Bone morphogenetic protein-2 (BMP2) signaling to the Col 2α1 gene in chondroblasts requires another Dlx homeobox gene, Dlx2 [55]. It has also been demonstrated that Dlx5 also regulates chondrocyte maturation by promoting conversion of immature proliferating chondrocytes into hypertrophic chondrocytes [35]. In conclusion, as established for early development [56], mutual interactions between Dlx homeoprotein and other homeoproteins [51,57] may control the site-specific transcriptional activity of differentiating and differentiated skeletogenic cells associated with the genetically programmed growth process. We provide new information about Dlx3 expression in various tissues and at different stages of cellular maturation. Future studies could further analyze the role of Dlx3 in the modulation of master genes in various cells, particularly the switch between hypertrophic chondrocytes and osteoblasts during endochondral ossification, and the terminal differentiation of dental cells.
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Acknowledgments Maria Morasso, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland, USA is acknowledged for Dlx3 antibodies. Samir Boukottaya (Faculté de Médecine Dentaire de Monastir) and Anthony Saul (Paris), are acknowledged for English assistance. Funding sources supporting the study: INSERM ATC vieillissement, Fondation Benjamin Delessert.
References [1] Feledy JA, Morasso MI, Jang SI, Sargent TD. Transcriptional activation by the homeodomain protein distal-less 3. Nucleic Acids Res 1999;27(3):764–70. [2] Panganiban G, Rubenstein JL. Developmental functions of the distalless/Dlx homeobox genes. Development 2002;129(19):4371–86. [3] Ghanem N, Jarinova O, Amores A, Long Q, Hatch G, Park BK, et al. Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters. Genome Res 2003;13:533–43. [4] Sumiyama K, Ruddle FH. Regulation of Dlx3 gene expression in visceral arches by evolutionarily conserved enhancer elements. Proc Natl Acad Sci U S A 2003;100(7):4030–4. [5] Shirasawa T, Sakamoto K, Takahashi H. Molecular cloning and evolutional analysis of a mammalian homologue of the distal-less (Dlx3) homeobox gene. FEBS Lett 1994;351:380–4. [6] Morasso MI, Markova NG, Sargent TD. Regulation of epidermal differentiation by a Distal-less homeodomain gene. J Cell Biol 1996;135:1879–87. [7] Anderson SA, Eisenstat DD, Shi L, Rubenstein JL. Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 1997;278(5337):474–6. [8] Qiu M, Bulfone A, Ghattas I, Meneses JJ, Christensen L, Sharpe PT, et al. Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev Biol 1997;185(2): 165–84. [9] Bendall AJ, Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development. Gene 2000;247(1–2):17–31. [10] Merlo GR, Zerega B, Paleari L, Trombino S, Mantero S, Levi G. Multiple functions of genes. Int J Dev Biol 2000;44:619–26. [11] Robinson GW, Mahon KA. Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development. Mech Dev 1994;48 (3):199–215. [12] Weiss KM, Bollekens J, Ruddle FH, Takashita K. Distal-less and other homeobox genes in the development of the dentition. J Exp Zool 1994;270(3):273–84. [13] Weiss KM, Ruddle FH, Bollekens J. Dlx and other homeobox genes in the morphological development of the dentition. Connect Tissue Res 1995;32(1–4):35–40. [14] Cobourne MT, Sharpe PT. Tooth and jaw: molecular mechanisms of patterning in the first branchial arch. Arch Oral Biol 2003;48(1):1–14. [15] Robledo RF, Rajan L, Li X, Lufkin T. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev 2002;16(9):1089–101. [16] Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione MP, Mantero S, et al. Craniofacial vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. Development 1999;126 (17):3795–809. [17] Depew CL, Liu JK, Long JE, Presley R, Meneses JJ, Pedersen RA, et
808
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
S. Ghoul-Mazgar et al. / Bone 37 (2005) 799–809 al. Dlx5 regulated regional development of the branchial arches and sensory capsules. Development 1999;126(17):3831–46. Ryoo HM, Hoffmann HM, Beumer T, Frenkel B, Towler DA, Stein GS, et al. Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol Endocrinol 1997;11(11):1681–94. Shea CM, Edgar CM, Einhorn TA, Gerstenfeld LC. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis. J Cell Biochem 2003;90(6):1112–27. Davideau JL, Demri P, Hotton D, Gu TT, MacDougall M, Sharpe P, et al. Comparative study of MSX-2, DLX-5, and DLX-7 gene expression during early human tooth development. Pediatr Res 1999; 46(6):650–6. Zhao Z, Stock D, Buchanan A, Weiss K. Expression of Dlx genes during the development of the murine dentition. Dev Genes Evol 2000;210(5):270–5. Masuda Y, Sasaki A, Shibuya H, Ueno N, Ikeda K, Watanabe K. Dlxin1, a novel protein that binds Dlx5 and regulates its transcriptional function. J Biol Chem 2001;276(7):5331–8. Lezot F, Thomas B, Hotton D, Forest N, Orestes-Cardoso S, Robert B, et al. Biomineralization, life-time of odontogenic cells and differential expression of the two homeobox genes MSX-1 and DLX-2 in transgenic mice. J Bone Miner Res 2000;15(3):430–41. Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, Wijnen AJV, et al. Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3 and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 2004;24 (20):9248–61. Park GT, Morasso MI. Regulation of the Dlx3 homeobox gene upon differentiation of mouse keratinocytes. J Biol Chem 1999;274 (37):26599–608. Morasso MI, Grinberg A, Robinson G, Sargent TD, Mahon KA. Placental failure in mice lacking the homeobox gene Dlx3. Proc Natl Acad Sci U S A 1999;96(1):162–7. Nissen RM, Yan J, Amsterdam A, Hopkins N, Burgess SM. Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning. Development 2003;130 (11):2543–54. Miller CT, Yelon D, Stainier DY, Kimmel CB. Two endothelin 1 effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Development 2003;130(7):1353–65. Miller CT, Schilling TF, Lee K, Parker J, Kimmel CB. Sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development. Development 2001;27(17):3815–28. Ivey K, Tyson B, Ukidwe P, McFadden DG, Levi G, Olson EN, et al. Galphaq and Galpha11 proteins mediate endothelin-1 signaling in neural crest-derived pharyngeal arch mesenchyme. Dev Biol 2003;255 (2):230–7. Price JA, Bowden DW, Wright JT, Pettenati MJ, Hart TC. Identification of a mutation in DLX3 associated with tricho-dentoosseous (TDO) syndrome. Hum Mol Genet 1998(3):563–9. Wright JT, Kula K, Hall K, Simmons JH, Hart TC. Analysis of the tricho-dento-osseous syndrome genotype and phenotype. Am J Med Genet 1997;72(2):197–204. Haldeman RJ, Cooper LF, Hart TC, Philips C, Boyd C, Wright T, et al. Increased bone density associated with Dlx3 mutation in the trichodento-osseous syndrome. Bone 2004;35:988–97. Marijanovic I, Kronenberg MS, Erceg I, Stover ML, Upholt WB, Lichtler AC. Dlx3, 5 and 6 may have redundant effects on bone development. JBMR 2002;17:M216. Ferrari D, Kosher RA. Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev Biol 2002;252 (2):257–70. Papagerakis P, Mac Dougall M, Hotton D, Bailleul-Forestier I, Oboeuf M, Berdal A. Expression of amelogenin in odontoblasts. Bone 2003;43 (2–3):509–14. Nefussi JR, Brami G, Modrowski D, Oboeuf M, Forest N. Sequential
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46] [47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
expression of bone matrix proteins during rat calvaria osteoblast differentiation and bone nodule formation in vitro. J Histochem Cytochem 1997;45(4):493–503. Sautier JM, Nefussi JR, Forest N. In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells. Biol Cell 1993;78(3):181–9. Davideau JL, Papagerakis P, Hotton D, Lezot F, Berdal A. In situ investigation of vitamin D receptor, alkaline phosphatase, and osteocalcin gene expression in oro-facial mineralized tissues. Endocrinology 1996;137(8):3577–85. Hotton D, Davideau JL, Bernaudin JF, Berdal A. In situ hybridization of calbindin-D28 k transcripts in undecalcified sections of the rat continuously erupting incisor. Connect Tissue Res 1995;32(1–4): 137–43. Bryan JT, Morasso MI. The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1. J Cell Sci 2000;113(22):4013–23. Balaton AL, Coindre JM, Collin F, Ettore F, Fiche M, Jacquemier J, et al. Recommendations for the immunohistochemical evaluation of hormone receptors on paraffin sections of breast cancer. Study Group on Hormone Receptors using Immunohistochemistry FNCLCC/ AFAQAP. National Federation of Centres to Combat Cancer/French Association for Quality Assurance in Pathology. Ann Pathol 1996;16 (2):144–8. Kergosien N, Sautier J-M, Forest N. Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system. Calcif Tissue Int 1998;62:114–21. Asselin A, Hattar S, Oboeuf M, Greenspan D, Berdal A, Sautier J-M. The modulation of tissue-specific gene expression in rat nasal chondrocyte cultures by bioactive glasses. Biomaterials 2004;25 (25):5621–30. Hotton D, Moreau N, Lézot F, Forest N, Berdal A. Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo. J Histochem Cytochem 1999;47(12):1541–52. Wurtz T, Berdal A. Osteoblasts precursors at different anatomic sites. Crit Rev Eukaryot Gene Expr 2003;13(2–4):147–61. Harris SE, Guo D, Harris MA, Krishnaswamy A, Lichtler A. Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors. Front Biosci 2003;8:1249–65. Shirakabe K, Terasawa K, Miyama K, Shibuya H, Nishida E. Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5. Genes Cells 2001;6(10): 851–6. Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM. BMP-2-induced osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 2003;309(3):689–94. Drissi MH, Li X, Sheu TJ, Zuscik MJ, Schwarz EM, Puzas JE, et al. Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes. J Cell Biochem 2003;90(6):1287–98. Blin-Wakkach C, Lezot F, Ghoul-Mazgar S, Hotton D, Monteiro S, Teillaud C, et al. Endogenous Msx1 antisense transcript: in vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals. Proc Natl Acad Sci 2001;98(13): 7336–41. Gibson CW, Yuan ZA, Hall B, Longenecker G, Chen E, Thyagarajan T, et al. Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem 2001;276(34):31871–5. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signalling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13(16): 2072–86. Chung U, Schipan E, Mac Mahon AP, Kronenberg H. Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 2001;107(3):295–304.
S. Ghoul-Mazgar et al. / Bone 37 (2005) 799–809 [55] Xu SC, Harris MA, Rubenstein JL, Mundy GR, Harris SE. Bone morphogenetic protein-2 (BMP-2) signalling to the Co2 alpha1 gene in chondroblasts requires the homeobox gene Dlx2. DNA Cell Biol 2001;20(6):359–65. [56] Francis-West P, Ladher R, Barlow A, Graveson A. Signalling
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interactions during facial development. Mech Dev 1997;75(1–2): 3–28. [57] Zhang H, Hu G, Wang H, Sciavolino P, Iler N, Shen MM, et al. Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism. Mol Cell Biol 1997;17(5):2920–32.
Expression pattern of Dlx3 during cell differentiation in mineralized tissues Sonia Ghoul-Mazgar a,b , Dominique Hotton a , Frédéric Lézot a , Claudine Blin-Wakkach c , Audrey Asselin a , Jean-Michel Sautier a , Ariane Berdal a,⁎ a
Laboratoire de Biologie Oro-faciale et Pathologie INSERM U 714, 15-21 rue de l'Ecole de Médecine 75270, Universités Paris 7 and Paris 6-IFR58, Paris Cedex 06, France b Laboratoire d'Histologie-Embryologie, Faculté de Médecine Dentaire de Monastir, Tunisia, France c Génétique et pathologies moléculaires CNRS-FRE 2720 Faculté de Médecine de Nice, Université Sophia-Antipolis, Nice, France Received 9 September 2004; revised 21 March 2005; accepted 30 March 2005 Available online 19 September 2005
Abstract The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1—its role in skeleton patterning, 2—its expression in dental epithelium and mesenchyme during morphogenesis, 3—the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4—recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid–osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton. © 2005 Elsevier Inc. All rights reserved. Keywords: Dlx; Enamel; Dentin; Cartilage; Bone
Introduction The Dlx gene family, related to Distal-less homeobox gene in Drosophila, encodes for proteins that act as transcription activators [1,2]. In vertebrates, there are six Dlx genes arranged in three clusters (Dlx1/Dlx2, Dlx3/ Dlx4 and Dlx5/Dlx6) [3]. Paired genes are localized on three distinct chromosomes with an inverted configuration and are separated by a short intergenic region [3,4]. ⁎ Corresponding author. Fax: +33 1 44 07 14 21. E-mail address: [email protected] (A. Berdal). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.03.020
Sharing cis-regulatory elements between members of Dlx bigene cluster may contribute to an observed overlap in gene expression pattern and a partial functional redundancy [5]. Dlx genes are key-players in development [6–10]. They are expressed in overlapping but distinct domains, primarily in the forebrain, branchial arches, sensory organs, limbs and tissues derived from epithelial–mesenchymal interactions [9,11]. In particular, Dlx genes are involved in early vertebrate morphogenesis of the head and dentition [12– 14]. Dlx homeobox genes have also been shown to be expressed during the morphogenesis of the distal regions of
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extending appendages which involves bone anlage formation [2,15]. The contribution of Dlx genes to skeletal morphogenesis has been established by the phenotypes of their invalidation in transgenic mice. Double Dlx1/Dlx2 null mutants exhibit major and site-specific defects in teeth and other craniofacial skeletal units [8]. Double targeted inactivation of Dlx5 and Dlx6 genes results in severe craniofacial, axial and appendicular skeletal abnormalities leading to perinatal lethality [15]. Non-redundant functions of Dlx homeoproteins have been documented in Dlx5−/− where a delay in osteoblast differentiation is observed [16,17]. Dlx genes have also been postulated to regulate cell differentiation in the skeleton including bone [18], cartilage [19] and tooth [20,21]. Compared to developmental studies, relatively few studies have focused on Dlx genes and proteins in the later process of biomineralization either in vitro [18,22] or in vivo [23]. Considerable attention has been paid to Dlx2 and Dlx5 and, very recently, during revision of this article, to Dlx3 [24]. Dlx3, a member of the Dlx family, is a transcription factor described in the neural crest and implicated during development of epithelial tissues such as skin, hair follicles, otic and olfactory placodes, limb bud and tooth germ. In epidermis, Dlx3 homeoprotein triggers the terminal differentiation of keratinocytes [25]. Gene-targeted mice are available, but their skeleton cannot be studied as loss of Dlx3 function is associated with lethal development due to its role in the placenta [26]. However, disturbed expression of Dlx3 is associated with skeletal defects observed in several mutant mice, inactivated for other genes (foo, [27], Hand2, [28]). Notably, the endothelin-1 signaling pathway has been well characterized and modulates Dlx3 expression (sucker mutation, [29], edn1downstream targets, [28]; Galphaq- and Galpha11-deficient embryos, [30]). The presence of Dlx3 mRNA in tooth germ has been reported during the bud, cap and bell stages of development ([11,21], this study). In humans, disruption of the Dlx3 coding sequence is associated with an inherited autosomal dominant disorder, Tricho–Dento–Osseous syndrome (TDOS). Despite the fact that the observed defects involve not only ectodermal derivatives, but also bone and teeth [31,32], little is known about this transcription factor in the normal skeleton and more specifically during cell differentiation and mineralization of extracellular matrices. TDOS is associated with defects in the craniofacial skeleton, which is essentially formed by a membranous bone formation process [32]. Furthermore, the detection of altered axial skeleton osteogenesis and the observed high mineral density in axial bone of known TDO individuals have recently suggested a role of Dlx3 in endochondral ossification [33]. Based on (1) the importance of Dlx3 during skeletal patterning [34], (2) the phenotype of TDOS related to Dlx3 mutations and (3) the emerging role of Dlx
homeoproteins in bone formation [35], the present study was designed to explore Dlx3 expression patterns in mineralized tissues in vitro and in vivo. The diverse secretory cells of mineralized tissues were shown to systematically express Dlx3. This investigation was made possible by the availability of differentiated cells from several sources according to the tissue-type: sampling of dental cells by microdissection of murine mandibular incisors, a classical experimental model system used to study the regulation of gene expression in ameloblasts and odontoblasts [36] and primary culture of craniofacial skeletogenic cells: calvarium osteoblasts for membranous bone formation [37] and nasal septum chondrocytes for endochondral bone formation [38]. During revision of this article, an in vitro study reported the direct role of Dlx3 in bone formation and upregulation of bone-related genes during osteoblast differentiation, via either protein–DNA or protein–protein interactions [24]. Our data show that, in vivo, the temporal window of Dlx3 homeoprotein expression varies specifically according to the terminal differentiation for each cell-type studied: ameloblasts, odontoblasts, osteoblasts and chondrocytes.
Materials and methods Preparation and characterization of the human Dlx3 probe Human samples A limited number of antenatal craniofacial tissues, excluding brain, were collected at Robert Debré hospital according to French ethical rules, and were used for total RNA extraction and generation of a Dlx3 riboprobe. Some samples from 9-week-old human embryos were also used for in situ hybridization [39,40]. Construction of the Dlx3 probe Two μg of total RNAs prepared using TRI-REAGENT protocol (Euromedex, Strasbourg, France) were reversetranscribed with an oligo(dT) primer according to the manufacturer's instructions (Invitrogen, San Diego, CA). Double-strand cDNA was amplified by a PCR with a regime of 94°C, 30 s (denaturing), 55°C, 30 s (annealing) and 72°C, 7 min (extension) for 35 cycles using sense P1dlx3 5′AAGGTCCGAAAGCCGCGTA-3′ and antisense P2dlx3 5′-CTGCTGCTGTAAGTGGGGT-3 (Genbank access number: NM-005220). The expected size (414 bp) for amplified PCR fragments was controlled on 2% agarose gel. The larger expected size (1947 bp including intron 2) of Dlx3 genomic DNA allowed verification of the absence of DNA contaminations in RNA extracts. Control PCR was also performed without reverse transcription, in order to exclude DNA contamination in RNA extracts. The amplified fragments were isolated and subcloned into pCR 2.1 plasmid (Invitrogen). In-frame cloning was confirmed by sequencing (Genome express, France).
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Northern blotting Ten μg of total RNA isolated from a 9-week-old embryo was fractionated electrophoretically into a 1% agarose formaldehyde gel and transferred onto nylon membranes (Amersham, Les Ulis, France). Membranes were successively prehybridized and then hybridized with antisense Dlx3 probes 32P-labeled by random priming (RediprimeII, Amersham Pharmacia Biotech, France) synthesized from pCR 2.1 plasmid after linearization with BamH1 restriction endonuclease and using T7 RNA polymerase (Gibco BRL, Life Technologies, Cergy-Pontoise, France). The hybridized blots were autoradiographed using Kodak films (Amersham).
Preparation of dental and chondrogenic cells for RT-PCR analysis
Investigation of in vivo Dlx3 expression
Cell culture method Chondrocytes were enzymatically isolated from the nasal septum of 21-day-old fetal Sprague–Dawley rats, as
Animals C57 Bl6 mice (Charles River Breeding Laboratories, France) were studied from Theiler developmental stage E14.5 to 1 day post-natally. Embryos were fixed with 4% paraformaldehyde immersion and cryostat sectioned [39,40]. Some embryos of the same developmental stage were also paraffin-embedded for immunohistochemistry [41]. In situ hybridization The Dlx3 antisense RNA digoxigenin-labeled probe (414 bp) was synthesized as described for the Northern blotting but using digoxigenin-UTP (Boehringer-Mannheim, Meylan, France). In situ hybridization was performed as previously described [39,40]: cryostat sections were hybridized with 30 μl of digoxigeninlabeled probes diluted to 1:200, and the reaction was revealed by anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche Diagnostics, Meylan, France). Sections were dehydrated, mounted under a coverslip and photographed. Immunohistochemistry Anti-Dlx3 polyclonal antiserum raised against a 16amino-acid synthetic peptide (aminoacids 242 to 256) of the murine Dlx3 protein (kindly provided by Maria Morasso, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland, USA; described in [41]) was used. Sections were deparaffinized, rehydrated and treated with citrate buffer (10 mM, pH 6) in the Microwave [42]. Sections were washed with 0.1 M phosphate-buffered saline-pH 7.3 and treated for 5 min with H2O2 in methanol then incubated for 1 h with the primary antibody and, after rinsing, were incubated for 10 min with streptavidin–peroxidase conjugated secondary antibodies (Amersham, France). The immune complexes were detected by a diaminobenzidine staining kit (DAKO Corporation, Carpinteria, CA, USA). Non-relevant rabbit immunoglobulins were used as negative control.
Microdissections Mandibles and alveolar bone of 56-day-old male C57Bl6 mice were removed under a dissecting microscope. The incisor was treated as follows: an apical portion containing the odontogenic organ and early mineralization zone of enamel and dentin was excluded in order to avoid epithelial– mesenchymal contamination. Lateral cuts were made along the cemento-enamel junction and the enamel organ was scraped from the labial part of the incisor. Finally, after crack opening the dentin, the dental mesenchyme was collected.
Table 1 Primers for RT-PCR assays Gene
Species
Size (bp)
Oligonucleotides
Dlx3
Rat
404
Mouse
414
Dlx2
Mouse
430
OC
Mouse
257
Amg
Mouse
370
ALP
Mouse
525
Calbindin D-28k
Mouse
480
GAPDH
Mouse
824
Rat
401
Rat
213
Sense primer: 5′-AAGTTCGTAAACCCAGGAC-3′ Antisense primer GGTGGGAATTGATTGAGCT Sense primer: 5′-AAGGTCCGAAAGCCGCGTA-3′ Antisense primer: 5′-CTGCTGCTGTAAGTGGGGT-3′ Sense primer: 5′-TCCTACCAGTACCAAGCCA-3′ Antisense primer: 5′-AAGCACAAGGTGGAGAAGC-3′ Sense primer: 5′-CTCACTCTGCTGGCCCTG-3′ Antisense primer: 5′-CCGTAGATGCGTTTGTAGGC-3′ Sense primer: 5′-GGATCAAGCATCCCTGAGCTTCAG-3′ Antisense primer: 5′-ATCATTGGTTGCTGGGGGCGCAC-3′ Sense primer: 5′-CATCTGGAACCGCACGGAAC-3′ Antisense primer: 5′-GCCTGGTAGTTGTTGTGAGC-3′ Sense primer: 5′-ACGGAAGTGGTTACCTGGAA-3′ Antisense primer: 5′-CACACATTTTGATTCCCTGG-3′ Sense primer: 5′-TTCCAGTATGATTCCACTCA-3′ Antisense primer: 5′-CTGTAGCCATATTCATTGTC-3′ Sense primer: 5′-GACCCCTTCATTGACCTCAACTA-3′ Antisense primer: 5′-AAGTTGTCATGGATGACCTTGGC-3′ Sense primer: 5′-GTCTCTTGCTAGAAGAGA-3′ Antisense primer: 5′-GCCTGCAGGGAAGGTCAT-3′
Ihh
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previously described [38,43,44]. Briefly, nasal septum was aseptically dissected and fragments were incubated in phosphate buffered solution with collagenase (400 U/ml) and hyaluronidase (750 U/ml) for 2 h at 37°C. Cells were then dissociated from the cartilage fragments and plated at 3.5 × 104 cell/cm2. The culture medium used was Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen), 10 mM β-glycerophosphate (Sigma), 50 μg/ml ascorbic acid (Sigma) and 50 U/ml of penicillin-streptomycin (Invitrogen). Cells were cultured for up to 21 days in a humidified atmosphere of 5% CO2 in air at 37°C. Culture media were changed 24 h after seeding and at 48-h intervals thereafter. This model has been established to represent the process of endochondral bone formation [38,43,44]. RT-PCR and real-time PCR analysis of murine tissues and cells As described for human cells (see previous chapter on construction of the Dlx3 probe), total RNAs were extracted,
reverse -transcribed and PCR was performed for 30 cycles. Table 1 summarizes the primers used, the sizes of the expected amplicons and the temperatures used. Real-time PCR was performed for Dlx3 and Indian Hedgehog (Ihh), using a Light cycler™ (Roche Diagnostic). Real-time RTPCR using SYBR Green I reaction was performed by a standard protocol. 2.5 μl of cDNA diluted at 1/100 was added to a 7.5-μl reaction mixture including MgCl2 at the optimal concentration (3–5 mM), each primer at the optimal concentration (4, 6 or 8 μm) and 1 μl of a Light cycler-DNA Master SYBR Green I (Roche Diagnostics) and incubated in a Light cycler ™ under the following conditions: at 95°C for 10 min as the denaturation program, followed by 40 or 50 cycles of 95°C for 10 s, each at the optimal annealing temperature for 10 s, 72°C for 10 s. The optimal conditions for each gene amplification and quantification are summarized for underlined primers in Table 1. The fluorescence measurement was recorded at the end of each cycle.
Fig. 1. Construction and validation of the Dlx3 probe. DLX3 probe was synthesized from a 9-week-old human embryo. The double-strand cDNA obtained was detected on 2% agarose gel, at the expected size of 414 bp (A). The absence of contamination with genomic DNA was confirmed by the absence of a 1947-bp amplicon. PCR performed without previous reverse transcription also demonstrates the absence of PCR amplification products (data not shown). Total cellular RNA (10 μg) from a 9-week-old human embryo was loaded onto a 1% formaldehyde agarose gel and hybridized with Dlx3 probe for Northern analysis (B). In situ hybridization using RNA digoxigenin-labeled probes on 9-week-old human embryo serial cryostat sections showed Dlx3-positive structures identified during bud (C) and cup stages (D) of dental development and in the palatal bone process (E).
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Results Generation and validation of the Dlx3 probe during human antenatal development (Fig. 1) 414 bp PCR amplicons were obtained from human samples (Fig. 1A), and used as probes by cloning in pCR2.1 plasmid. Antisense riboprobes allowed visualization of the Dlx3 transcript (≈4500 bp) in human orofacial samples from 9-week-old embryos (Fig. 1B). In situ hybridization was also performed with the same riboprobe in human samples (Figs. 1C, D and E). Epithelial cells of tooth bud (Fig. 1C) and cap stages (Fig. 1D) contained Dlx3 RNAs. Ossification centers in the maxilla were also Dlx3-positive (Fig. 1E). Distribution of Dlx3 mRNA and protein during tooth morphogenesis and cell differentiation in mice (Fig. 2) In view of the ethical limitations on human studies, Dlx3 expression was more systematically analyzed in mice. Data on epidermis (non shown) were identical to the results of other published studies [41]. As previously shown by in situ hybridization [21], Dlx3 expression was consistently observed at the mRNA (Fig. 2A) and protein levels (Fig. 2B) on serial sections of first molar tooth germs at Theiler stage embryonic day 15.5 (E15.5) in mice. The relative expression levels of Dlx3 transcripts were still high
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at the later bell stage of the first molar of 1-day-old mice (Fig. 2C), but were different in dental epithelium and mesenchyme (Fig. 2D). Dlx3 transcripts appeared to be regularly expressed in differentiating and differentiated ameloblasts (Fig. 2C), but were down-regulated during odontoblast polarization (Fig. 2D). Dlx3 expression was also analyzed by RT-PCR in microdissected murine dental cells (Fig. 2E). Mouse incisors were used for this study, as they are an established model for RNA investigation. The entire developmental sequence of amelogenesis and dentinogenesis is recapitulated in this experimental model system and distributed along the labial aspect of the mandibular incisor. The size of these continuously erupting teeth allows microscopic dissection of epithelial (forming enamel) and mesenchymal (forming dentin) cells. The amplicons shown in figure F illustrate the absence of epithelial–mesenchymal cross-contaminations during microdissection procedures, as established by several studies. Some of these genes are co-expressed in mesenchyme and epithelium (calbindinD-28k, alkaline phosphatase, and amelogenin), as established in previous reports [36,40, 45]). In contrast, Dlx2 homeogene, which is exclusively present in the epithelial compartment [23], was only detected in microdissected epithelium. Conversely, osteocalcin which is exclusively synthesized by odontoblasts, showed amplified cDNA exclusively in the corresponding mesenchyme. This method of validation confirmed co-
Fig. 2. In vivo investigation of Dlx3 transcript and protein in dental tissues. On serial sections of first molar tooth germs at Theiler stage 15.5, Dlx3 transcripts (A) and protein (B) were both detected in dental epithelium and mesenchyme. This expression persisted until the later bell stage observed 1 day post-natally (C). The highest magnification of the box in C shows that, in preodontoblasts (pod) and in undifferentiated ameloblasts (Am), transcripts were strongly expressed (D). However, in differentiated and polarized odontoblasts (od), this expression was absent. Phenotypic transcripts expressed either in mice microdissected dental mesenchyme (M) or epithelium (Ep) were analyzed by RT-PCR (F). Agarose gel analysis showed that Dlx3, ALP, Amg and calbindin 28-k were detected in both tissues, whereas Dlx2 was only detected in dental epithelium and OC, only in dental mesenchyme. GAPDH levels served as a loading control.
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expression of Dlx3 in both the mesenchymal and epithelial compartments.
Dlx3 protein expression during endochondral ossification (Fig. 4)
Dlx3 protein expression during intramembranous bone formation (Fig. 3)
Dlx3 protein expression was observed in the five classically described zones in the growth plate cartilage, as exemplified in murine ribs at Theiler stage E15.5 (Fig. 4F). Dlx3 protein was not detected in the resting zone (Fig. 4A). In the proliferative zone, Dlx3 protein was detected (Fig. 4B) and strongly expressed in the pre-hypertrophic cartilage zone where chondrocytes became much larger (Fig. 4C). No Dlx3 protein was detected in the calcified cartilage zone (Fig. 4D). Bone collar produced in the deep portions of the perichondrium surrounding the cartilage, was also analyzed. The newly differentiated periosteum covering newly deposited bone formed a continuous layer of Dlx3-positive osteoblasts over bone collar (Fig. 4D). A comparative study of various long bones in the autopodium and ribs demonstrated two distinct steps in growth plate cartilages, as shown in Figs. 4G to J. Figs. 4G and H illustrate the absence of Dlx3 labeling in the perichondrium, i.e., in the future bone collar and periosteum,
Frontal sections of the palate visualized Dlx3 labeling at the site of initiation of membranous ossification (Fig. 3A). Serial sections detected Dlx3 protein at sites of mesenchymal condensation (Fig. 3B) and in bone trabeculae containing positive cells (Fig. 3C). At the same stage of development, in situ hybridization showed a similar distribution for Dlx3 transcripts in relation to cell differentiation from E14.5 to 1day-post-natally (Fig. 3D). In forming alveolar bone, Dlx3 transcripts were clearly localized in cells lining bone trabeculae (Fig. 3E). Differentiating (Fig. 3F) and differentiated osteoblasts (Fig. 3G) also appeared to express Dlx3 homeoprotein. Finally, the recently embedded osteocyte, also called osteoid–osteocyte, contained Dlx3 RNAs, while more mature osteocytes were negative in mice and even in human samples (Fig. 3H).
Fig. 3. Immunohistochemical detection of Dlx3 protein and in situ hybridization of Dlx3 at Theiler stage 15.5 in mouse intramembranous bone. During palatal (p) membranous ossification process (A), serial frontal sections of Theiler stage E15.5 mouse embryos showed that Dlx3 protein was detected in the cell condensation (B) and the osteoblast lining bone trabeculae (t) of the maxilla (C). Transcripts were also detected in the osteoblast lining trabeculae (t) (D). Later, in 1-day-old mice, Dlx3 transcript expression was also detected in osteoblasts lining alveolar bone trabeculae (t) (E). Osteoblast progenitors (obp) (F), osteoblasts (ob) and newly differentiated osteocytes (noc) appeared to express Dlx3 protein (vertical arrows), whereas well differentiated osteocytes (oc) (horizontal arrows) did not (G). During palatal membranous ossification of a 9-week-old human embryo, Dlx3 transcripts were also detected in osteoblast progenitors (obp), osteoblasts (ob) and newly differentiated osteocytes (noc), but were absent in well-differentiated osteocytes (oc) (H). M: oral mesenchyme; OE: oral epithelium.
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Fig. 4. Immunohistochemical detection of Dlx3 protein at Theiler stage 16.5 in endochondral bone ossification centers. Growth plate cartilage accompanying endochondral bone ossification was observed on ribs (A–F and I–J) and fingers (G–H). Dlx3 protein was absent from the resting zone (A ×1000), but was observed in the proliferative zone (B ×1000), strongly expressed in the pre-hypertrophic chondrocytes (C ×1000) and was not detectable in the hypertrophic and calcified cartilage zones (D ×1000). Bone collar presented a continuous layer of Dlx3 positive osteoblasts under the periosteum (E ×1000). Underneath the perichondrium that covers younger cartilage, only prehypertrophic cells expressed Dlx3 (G ×400 and H ×1000). Osteoblasts of newly-formed bone collar (I ×1000) and the central primary ossification point (J ×1000) also expressed Dlx3 protein.
before osteoblast differentiation. In contrast, Fig. 4I shows immunopositive osteoblasts of the newly formed bone collar and Fig. 4J shows similar appearance in the central primary ossification point.
was used for biomaterial investigation [44]. Ihh (Fig. 5E) showed a distinct upregulation with a maximal level at day 12. Interestingly, Dlx3 showed the same modulation pattern of expression levels as Ihh (Fig. 5E).
RT-PCR of Dlx3 and Ihh cDNA in nasal septum cartilage cells (Fig. 5)
Discussion
In primary cultures, well differentiated osteoblasts obtained from calvarium (data non shown) and rat nasal septum chondrocytes (Fig. 5) appeared to express Dlx3 transcripts. Rat nasal septum culture can be used to assess the chondrocyte life-cycle during the process of endochondral bone formation. Several stages are distinguished: early stages of mesenchymal precartilaginous condensation (Fig. 5A), differentiation of chondrocytes (Figs. 5B and C) and finally extracellular biomineralization (Fig. 5D). This system can also be used to characterize molecular events related to chondrocyte cell stages by real-time RT-PCR showing types II and X collagens, aggrecan, Runx2 and Ihh expression and
This descriptive study focused on the expression pattern of Dlx3 homeoprotein and transcript in Dlx3 target-tissues, as illustrated by the TDOS phenotype. These tissues include: (i) epidermis and hair follicle, which have been extensively characterized in other studies [41], (ii) dental epithelium and mesenchyme [11,21] and (iii) forming bone, which have been analyzed previously [34] albeit in less detail. The initial step of this study was therefore designed to verify the existence of Dlx3 transcripts in the 4 skeletogenic cell types studied by RT-PCR. Interestingly, during revision of this manuscript, publication highlighted Dlx3 expression and function in osteoblasts [24]. Driven overexpression and
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Fig. 5. Analysis of Dlx3 and Ihh expression in primary cultures of rat chondrocytes. (A) Day 5 in culture: precartilaginous condensations are visible in the confluent cell layer. Precartilaginous condensations are indicated with white arrows. (B) Day 8 in culture: phase bright colonies of differentiating chondrocytes are present in the culture dish. (C) Day 12 in culture: differentiating areas appear to be more numerous in the cell layer. (D) Day 21 in culture: the differentiating areas are enlarged. Dark areas (stars) are visible corresponding to mineralization of the matrix. (E) Gene expression level (relative to GAPDH) of Dlx3 and Ihh were determined at days 5, 8, 12 and 21, using real-time PCR. The signaling molecule Ihh was strongly expressed at day 12 and a similar expression pattern was observed for Dlx3 transcript.
knock-down of Dlx3 in vitro established that Dlx3 controls the appearance of osteogenic markers [24]. The present study allowed characterization of Dlx3 mRNA in other mineralized tissues using various experimental model systems, for enamel (microdissected dental epithelium, [36]), dentin (microdissected dental mesenchyme; [36]) and endochondral bone formation (primary culture of nasal septum chondrocytes; [38]). The cDNA and antibodies used in this study were validated by the identity of our results and available data for Dlx3 mRNA or protein in tooth [11,21], osteoblasts [24] and epidermis [41], (this study, data non shown) [6]. In situ hybridization and immunolabeling identified Dlx3 in ameloblasts, differentiating odontoblasts, osteoblasts and finally chondrocytes. These findings therefore provide evidence that Dlx3 is not only expressed during early skeletal patterning, but also in all mineralized tissues studied, inside secretory cells and at later and distinct differentiation stages. While some functional redundancy for various Dlx homeoproteins has been illustrated using exclusive [16] and combined null mutations during early patterning [8,15] distinct Dlx homeoproteins have been shown in vitro to be instrumental in osteoblast [24,46–49] and chondrocyte [35,50] physiology. Dlx2 and Dlx5 are involved in the osteoinductive cascade of BMP2 [47]. In osteoblasts, these two homeoproteins as well as Dlx3 play a role in the transcriptional activity of osteocalcin gene and mediation of BMP2-induced Runx2 expression [48,49]. BMP2 has also
been shown to significantly increase Dlx3 expression during induced osteoblast differentiation [24]. Dlx3 protein stimulates osteocalcin promoter activity and Dlx3–Runx2 interaction inhibits the Runx-mediated transcription of osteocalcin gene [24]. During endochondral bone formation [35], Dlx5 appears to control chondrocyte maturation. This function has also been proposed in the odontoblast lineage, where (i) Dlx5 downregulation occurs during odontoblast terminal differentiation [20], similar to the downregulation of Dlx3 in our study, and (ii) a matrix protein expressed in differentiated odontoblasts, osteocalcin, is negatively regulated by Dlx5 [51]. The present study provides a comparative overview of Dlx3 involvement in various mineralized tissues, which raises the question of their significance in each tissuespecific context. In tooth formation, while Dlx3 was continuously expressed in differentiating and differentiated ameloblasts, it appeared to be down-regulated during odontoblast polarization. Consistently with the present data, dental anomalies associated with 4 bp deletion of human Dlx3 gene [31,32] consist of small, yellow-brown teeth with thin hypoplastic enamel (i.e. affected enamel secretion) and large taurodontic pulp chamber (i.e. affected dental pulp cell differentiation). Dlx3 therefore appears to control distinct formation stages during the process of odontogenesis depending on the tissue. In secretion stage ameloblasts, the Dlx3 pattern and enamel phenotype of TDOS suggest that
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Dlx3 controls enamel thickness. Enamel thickness is dependent on the secretion of the major enamel protein: amelogenin [52]. The presumed gene positively regulated by Dlx3 would therefore be the amelogenin gene. In secretion stage ameloblasts, a combinatorial role for Dlx homeoproteins may also be proposed, as quantitative studies have previously established an inverse relationship between Dlx2 expression levels and enamel thickness [23]. This type of developmental stage specification for Dlx3 involvement in ameloblasts and odontoblasts raises the question of its role in osteoblasts and chondrocytes during the respective processes of membranous and endochondral bone formation, as recent clinical investigation of TDOS defects have established that not only craniofacial [32], but also appendicular [33] skeleton formation is affected by DLX3 gene mutations. The appendicular skeleton is formed by the endochondral bone formation process which involves chondrocytes. In the present study, in the osteoblastic lineages, in contrast with odontoblasts where Dlx3 protein was down-regulated in terminally differentiated cells, Dlx3 protein was expressed from the preosteoblast stage until the osteoid–osteocyte stage. Down-regulation of Dlx3 protein was observed only in mature osteocytes. The present in vivo study provides a detailed description of Dlx3 protein expression levels in the osteoblast–osteocyte maturation process, which is complementary to recently published functional studies on Dlx3 in osteoblasts [24]. In endochondral ossification, Dlx3 protein appeared to be upregulated in prehypertrophic chondrocytes and its expression levels decreased in hypertrophic chondrocytes, in vivo and in vitro. Indian hedgehog (Ihh) is one of the main regulators of long bone development and like Dlx3, it is expressed by chondrocytes leaving the proliferative pool (prehypertrophic chondrocytes) [53]. Ihh couples chondrogenesis to osteogenesis in endochondral bone development, perhaps in synergy with BMP2 [54] and potentially Dlx3, as suggested here. Indeed, PCR data showed a parallel variation of Dlx3 and Ihh transcript levels which were maximal at day 12 in rat chondrocytes in vitro. Bone morphogenetic protein-2 (BMP2) signaling to the Col 2α1 gene in chondroblasts requires another Dlx homeobox gene, Dlx2 [55]. It has also been demonstrated that Dlx5 also regulates chondrocyte maturation by promoting conversion of immature proliferating chondrocytes into hypertrophic chondrocytes [35]. In conclusion, as established for early development [56], mutual interactions between Dlx homeoprotein and other homeoproteins [51,57] may control the site-specific transcriptional activity of differentiating and differentiated skeletogenic cells associated with the genetically programmed growth process. We provide new information about Dlx3 expression in various tissues and at different stages of cellular maturation. Future studies could further analyze the role of Dlx3 in the modulation of master genes in various cells, particularly the switch between hypertrophic chondrocytes and osteoblasts during endochondral ossification, and the terminal differentiation of dental cells.
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Acknowledgments Maria Morasso, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland, USA is acknowledged for Dlx3 antibodies. Samir Boukottaya (Faculté de Médecine Dentaire de Monastir) and Anthony Saul (Paris), are acknowledged for English assistance. Funding sources supporting the study: INSERM ATC vieillissement, Fondation Benjamin Delessert.
References [1] Feledy JA, Morasso MI, Jang SI, Sargent TD. Transcriptional activation by the homeodomain protein distal-less 3. Nucleic Acids Res 1999;27(3):764–70. [2] Panganiban G, Rubenstein JL. Developmental functions of the distalless/Dlx homeobox genes. Development 2002;129(19):4371–86. [3] Ghanem N, Jarinova O, Amores A, Long Q, Hatch G, Park BK, et al. Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters. Genome Res 2003;13:533–43. [4] Sumiyama K, Ruddle FH. Regulation of Dlx3 gene expression in visceral arches by evolutionarily conserved enhancer elements. Proc Natl Acad Sci U S A 2003;100(7):4030–4. [5] Shirasawa T, Sakamoto K, Takahashi H. Molecular cloning and evolutional analysis of a mammalian homologue of the distal-less (Dlx3) homeobox gene. FEBS Lett 1994;351:380–4. [6] Morasso MI, Markova NG, Sargent TD. Regulation of epidermal differentiation by a Distal-less homeodomain gene. J Cell Biol 1996;135:1879–87. [7] Anderson SA, Eisenstat DD, Shi L, Rubenstein JL. Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 1997;278(5337):474–6. [8] Qiu M, Bulfone A, Ghattas I, Meneses JJ, Christensen L, Sharpe PT, et al. Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev Biol 1997;185(2): 165–84. [9] Bendall AJ, Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development. Gene 2000;247(1–2):17–31. [10] Merlo GR, Zerega B, Paleari L, Trombino S, Mantero S, Levi G. Multiple functions of genes. Int J Dev Biol 2000;44:619–26. [11] Robinson GW, Mahon KA. Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development. Mech Dev 1994;48 (3):199–215. [12] Weiss KM, Bollekens J, Ruddle FH, Takashita K. Distal-less and other homeobox genes in the development of the dentition. J Exp Zool 1994;270(3):273–84. [13] Weiss KM, Ruddle FH, Bollekens J. Dlx and other homeobox genes in the morphological development of the dentition. Connect Tissue Res 1995;32(1–4):35–40. [14] Cobourne MT, Sharpe PT. Tooth and jaw: molecular mechanisms of patterning in the first branchial arch. Arch Oral Biol 2003;48(1):1–14. [15] Robledo RF, Rajan L, Li X, Lufkin T. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev 2002;16(9):1089–101. [16] Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione MP, Mantero S, et al. Craniofacial vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. Development 1999;126 (17):3795–809. [17] Depew CL, Liu JK, Long JE, Presley R, Meneses JJ, Pedersen RA, et
808
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
S. Ghoul-Mazgar et al. / Bone 37 (2005) 799–809 al. Dlx5 regulated regional development of the branchial arches and sensory capsules. Development 1999;126(17):3831–46. Ryoo HM, Hoffmann HM, Beumer T, Frenkel B, Towler DA, Stein GS, et al. Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol Endocrinol 1997;11(11):1681–94. Shea CM, Edgar CM, Einhorn TA, Gerstenfeld LC. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis. J Cell Biochem 2003;90(6):1112–27. Davideau JL, Demri P, Hotton D, Gu TT, MacDougall M, Sharpe P, et al. Comparative study of MSX-2, DLX-5, and DLX-7 gene expression during early human tooth development. Pediatr Res 1999; 46(6):650–6. Zhao Z, Stock D, Buchanan A, Weiss K. Expression of Dlx genes during the development of the murine dentition. Dev Genes Evol 2000;210(5):270–5. Masuda Y, Sasaki A, Shibuya H, Ueno N, Ikeda K, Watanabe K. Dlxin1, a novel protein that binds Dlx5 and regulates its transcriptional function. J Biol Chem 2001;276(7):5331–8. Lezot F, Thomas B, Hotton D, Forest N, Orestes-Cardoso S, Robert B, et al. Biomineralization, life-time of odontogenic cells and differential expression of the two homeobox genes MSX-1 and DLX-2 in transgenic mice. J Bone Miner Res 2000;15(3):430–41. Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, Wijnen AJV, et al. Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3 and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 2004;24 (20):9248–61. Park GT, Morasso MI. Regulation of the Dlx3 homeobox gene upon differentiation of mouse keratinocytes. J Biol Chem 1999;274 (37):26599–608. Morasso MI, Grinberg A, Robinson G, Sargent TD, Mahon KA. Placental failure in mice lacking the homeobox gene Dlx3. Proc Natl Acad Sci U S A 1999;96(1):162–7. Nissen RM, Yan J, Amsterdam A, Hopkins N, Burgess SM. Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning. Development 2003;130 (11):2543–54. Miller CT, Yelon D, Stainier DY, Kimmel CB. Two endothelin 1 effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Development 2003;130(7):1353–65. Miller CT, Schilling TF, Lee K, Parker J, Kimmel CB. Sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development. Development 2001;27(17):3815–28. Ivey K, Tyson B, Ukidwe P, McFadden DG, Levi G, Olson EN, et al. Galphaq and Galpha11 proteins mediate endothelin-1 signaling in neural crest-derived pharyngeal arch mesenchyme. Dev Biol 2003;255 (2):230–7. Price JA, Bowden DW, Wright JT, Pettenati MJ, Hart TC. Identification of a mutation in DLX3 associated with tricho-dentoosseous (TDO) syndrome. Hum Mol Genet 1998(3):563–9. Wright JT, Kula K, Hall K, Simmons JH, Hart TC. Analysis of the tricho-dento-osseous syndrome genotype and phenotype. Am J Med Genet 1997;72(2):197–204. Haldeman RJ, Cooper LF, Hart TC, Philips C, Boyd C, Wright T, et al. Increased bone density associated with Dlx3 mutation in the trichodento-osseous syndrome. Bone 2004;35:988–97. Marijanovic I, Kronenberg MS, Erceg I, Stover ML, Upholt WB, Lichtler AC. Dlx3, 5 and 6 may have redundant effects on bone development. JBMR 2002;17:M216. Ferrari D, Kosher RA. Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev Biol 2002;252 (2):257–70. Papagerakis P, Mac Dougall M, Hotton D, Bailleul-Forestier I, Oboeuf M, Berdal A. Expression of amelogenin in odontoblasts. Bone 2003;43 (2–3):509–14. Nefussi JR, Brami G, Modrowski D, Oboeuf M, Forest N. Sequential
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46] [47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
expression of bone matrix proteins during rat calvaria osteoblast differentiation and bone nodule formation in vitro. J Histochem Cytochem 1997;45(4):493–503. Sautier JM, Nefussi JR, Forest N. In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells. Biol Cell 1993;78(3):181–9. Davideau JL, Papagerakis P, Hotton D, Lezot F, Berdal A. In situ investigation of vitamin D receptor, alkaline phosphatase, and osteocalcin gene expression in oro-facial mineralized tissues. Endocrinology 1996;137(8):3577–85. Hotton D, Davideau JL, Bernaudin JF, Berdal A. In situ hybridization of calbindin-D28 k transcripts in undecalcified sections of the rat continuously erupting incisor. Connect Tissue Res 1995;32(1–4): 137–43. Bryan JT, Morasso MI. The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1. J Cell Sci 2000;113(22):4013–23. Balaton AL, Coindre JM, Collin F, Ettore F, Fiche M, Jacquemier J, et al. Recommendations for the immunohistochemical evaluation of hormone receptors on paraffin sections of breast cancer. Study Group on Hormone Receptors using Immunohistochemistry FNCLCC/ AFAQAP. National Federation of Centres to Combat Cancer/French Association for Quality Assurance in Pathology. Ann Pathol 1996;16 (2):144–8. Kergosien N, Sautier J-M, Forest N. Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system. Calcif Tissue Int 1998;62:114–21. Asselin A, Hattar S, Oboeuf M, Greenspan D, Berdal A, Sautier J-M. The modulation of tissue-specific gene expression in rat nasal chondrocyte cultures by bioactive glasses. Biomaterials 2004;25 (25):5621–30. Hotton D, Moreau N, Lézot F, Forest N, Berdal A. Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo. J Histochem Cytochem 1999;47(12):1541–52. Wurtz T, Berdal A. Osteoblasts precursors at different anatomic sites. Crit Rev Eukaryot Gene Expr 2003;13(2–4):147–61. Harris SE, Guo D, Harris MA, Krishnaswamy A, Lichtler A. Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors. Front Biosci 2003;8:1249–65. Shirakabe K, Terasawa K, Miyama K, Shibuya H, Nishida E. Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5. Genes Cells 2001;6(10): 851–6. Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM. BMP-2-induced osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 2003;309(3):689–94. Drissi MH, Li X, Sheu TJ, Zuscik MJ, Schwarz EM, Puzas JE, et al. Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes. J Cell Biochem 2003;90(6):1287–98. Blin-Wakkach C, Lezot F, Ghoul-Mazgar S, Hotton D, Monteiro S, Teillaud C, et al. Endogenous Msx1 antisense transcript: in vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals. Proc Natl Acad Sci 2001;98(13): 7336–41. Gibson CW, Yuan ZA, Hall B, Longenecker G, Chen E, Thyagarajan T, et al. Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem 2001;276(34):31871–5. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signalling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13(16): 2072–86. Chung U, Schipan E, Mac Mahon AP, Kronenberg H. Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 2001;107(3):295–304.
S. Ghoul-Mazgar et al. / Bone 37 (2005) 799–809 [55] Xu SC, Harris MA, Rubenstein JL, Mundy GR, Harris SE. Bone morphogenetic protein-2 (BMP-2) signalling to the Co2 alpha1 gene in chondroblasts requires the homeobox gene Dlx2. DNA Cell Biol 2001;20(6):359–65. [56] Francis-West P, Ladher R, Barlow A, Graveson A. Signalling
809
interactions during facial development. Mech Dev 1997;75(1–2): 3–28. [57] Zhang H, Hu G, Wang H, Sciavolino P, Iler N, Shen MM, et al. Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism. Mol Cell Biol 1997;17(5):2920–32.