Studies Of The Roles Of Twist1/2&Runx2in Bone And Tooth Development

IntroductionMammalian Twistl and Twist2are two members of the Twist subfamily of the basic-helix-loop-helix (bHLH) transcription factors that have been highly conserved during evolution. In Drosophila, a single Twist gene, DTwist, is essential for embryonic gastrulation and mesodermal formation. Mouse Twistl was identified by its high homology with DTwist, while Twist2, originally called "Dermol", was discovered by a yeast-two-hybrid screen using the ubiquitous bHLH protein E12as bait. The expression patterns of Twistl and Twist2show an extensive overlap during mouse embryonic development, and their encoded proteins exhibit a high degree (up to98%) of sequence similarity. Both proteins perform various functions by forming either homodimers or heterodimers with bHLH E proteins (E12/E47) that bind to DNA canonical regulatory sequences called "E-boxes"(CANNTG) in the promoter region of target genes.In humans, mutations in the TWIST1gene are associated with Saethre-Chotzen Syndrome (SCS), which is an autosomal dominant disorder characterized by craniosynostosis, brachydactyly, soft tissue syndactyly and facial dysmorphism. The skeletal phenotype of Twistl-heterozygous mouse consistently resembles that of human SCS with premature fusion of the cranial suture. As mouse embryonic development progresses, the Twistl expression declines in the developing bones of the skull. In addition, Twistl overexpression was found to inhibit osteoblast differentiation in vitro and in vivo. Together, these observations suggest that Twistl negatively regulates osteoblast differentiation and bone formation.Various molecular mechanisms may be responsible for the inhibitory role of Twist1in osteoblast differentiation. Twistl may modulate FGF signaling, especially Fgfr2expression in cranial suture development or it may directly bind to and inhibit the transactivation function of Runx2, a master regulator of osteogenesis. In addition, Twistl might indirectly regulate the Runx2expression through modulating FGFR2expression as shown in the ex vivo cultured primary osteoblasts isolated from human SCS patients. Finally, it is possible that Twistl inhibits osteoblast apoptosis via the suppression of TNF-α expression.Twist2has been shown to have an inhibitory function similar to that of Twistl in bone formation. While recessive TWIST2mutations in humans and its inactivation in mice result in a focal facial dermal dysplasia (FFDD) syndrome, there is no Twist1-deficient skeletal phenotype. The phenotypic difference between the Twistl-and Twist2-deficient subjects is indeed intriguing when viewed in the context of their significantly overlapping expression patterns in vivo and their similar functions in bone formation. Thus, it is largely unknown how Twistl and Twist2synergistically regulate bone formation and what molecular mechanism is involved.In this study, we generated a compound Twistl-and Twist2-haploinsufficient animal model, Twist1flox+; Twist2Cre/+mice, by crossing Twistl floxed mice with Twist2Cre knock-in mice. Thus, the compound Twist1flox+; Twist2Cre/+mice had one allele of Twist2replaced by the cre recombinase and one allele of Twist1deleted specifically in tissues where the Twist2gene was expressed. And then the Twistl-and Twist2-haploinsufficient mice were crossed to the Twist1flox/flox mice to obtain Twist1flox/flox, Twistcre/+mice.To our surprise, we found that the Twist1flox/+; Twist2Cre/+mice exhibited reduced bone formation and impaired proliferation and differentiation of osteoprogenitors. The bone formation in Twist1flox/flox, TwistCre/+mice were severely affected, as well as the tooth development. The skeletal and tooth abnormalities were associated with reduced FGF signaling as a consequence of the decreased expressions of several Fgf genes and Fgfrs.Objectives:1. To study the synergistical role of Twist1and Twist2in bone and tooth development.2. To study wheather Twistl and Twist2inhibit bone formation3. To study the cellular molecular mechanisms by which Twistl and Twist2regulate bone formation4. To study the role of Twistl and Twist2in tooth development and the possible mechanism.Materials and Methods:1. Generation of animal modelsThe Twist1floxflox mice were bred with Twist2cre+mice to generate compound Twist1flox+; Twist2cre+mice. And then the Twist1flox/+; Twist2cre+mice were crossed to the Twist1flox/flox mice to obtain Twist1flox/flox, Twistcre/+mice.2. Sample preparationsThe femurs and tibiae from one-week-old Twistlflox; Twist2crc+mice and control mice were dissected free of the skeletal muscles and fixed in4%paraformaldehyde, and then were proceeded directly for X-ray and micro-CT or Alcian blue/alizarin red staining. For histological staining, bone samples were decalcified in10%EDTA with0.5%paraformaldehyde, and embedded in paraffin using standard procedures. 3. Histology, immunohistochemistry and in situ hybridizationThe bone sections were then used for Hematoxylin and Eosin (H&E) staining, Tatrate-resistant alkaline phosphatase (TRAP) staining, immunohistochemistry or in situ hybridization.The following gene expressions were detected:Alp、Ocn、Dmp1、Osterix, Biglycan by using specific probes or antibodies/4. Cell proliferation assayOne-week-old Twist1flox/+; Twist2cre/+mice were intraperitoneally injected with5-bromo-2’-deoxyuridine (BrdU)(10mg/100g body weight) at24hours and then two hours before sacrifice. The long bones were collected and processed for paraffin sectioning as described above. The incorporated BrdU was detected with a BrdU staining kit (Invitrogen, Camarillo, CA, USA) according to the manufacturer’s instructions.5. Analysis of cell apoptosisApopTag(?) fluorescence In Situ Apoptosis Detection Kit (Chemicon(?), International, Inc) was applied to analyze cell apoptosis according to the manufacturer’s instruction. DAPI were used for nuclei staining.6. Quantitative Real-time PCRTotal RNA was extracted using Trizol from the long bones of the Twistl/2dHet mice and control mice. Tooth buds of Twist1flox/flox, Twistcre/+mice and control mice at E14.5were dissected under a stereomicroscope and the total RNA was extraxted. All RNA was reverse-transcribed into cDNA with a Reverse Transcription Kit. Quantitative real-time PCR (qPCR) was performed using a Go Tag(?) qPCR Master Mix System. Mouse18S rRNA primers were used for normalization.7. Cell culture, constructs and promoter luciferase assayThe C3H10T1/2mesenchymal cells, MC3T3-E1preosteoblast cells and MDPC-23odontoblast-like cells were cultured in required media and then were plated in24-well plates at a density of3×104cells per well. Then the cells were transiently transfected with0.1μg of4.9kb-Fgfr2promoter luciferase construct, together with0.4jig of various constructs expressing Twist1, Twist2or E12using FuGENE(?)6Transfection Reagent. Forty-eight hours later, the transfected cells were analyzed using a dual luciferase reporter assay system.Results:1. Twist1and Twist2deficient mice exhibit abnormal bone and tooth development. The alcian blue/alizarin red staining showed that Twist1flox+; Twist2Cre/+mice had a much smaller skeleton with delayed fusion of the interfrontal suture, open posterior fontanelles and delayed ossification in the metatarsals and phalanges. And the Twist1flox/flox, Twistcre/+mice exhibit even more severe skeletal and cranial bone formation. Plain X-radiography showed that the Twist1flox/+; Twist2Cre/+mice had reduced opacity in the tibiae compared to the control mice. Micro-CT images further confirmed that the Twist1flox/+; Twist2Cre/+mice had reduced trabecular bone and decreased cortical bone thickness. H&E staining revealed that the tooth bud development was retardant in the Twist1flox/flox) Twistcre/+mice and the cusp formation was abnormal.2. The osteoblast differentiation were impared in Twistl and Twist2double heterozygous mice.We examined the expression levels of the osteoblast differentiation markers by in situ hybridization, immunohistochemistry and real-time PCR. These methods revealed that the levels of the osteoblast differentiation markers Runx2, Osterix, Alp, Ocn, biglycan and Bsp were sharply reduced in the Twist1flox/+; Twist2Cre/+mice compared to the control mice. In addition, the osteocyte marker Dmpl was also dramatically decreased. However, TRAP staining showed that the number, distribution and size of the osteoclasts in the Twist1fox/+; Twist2Cre/+mice were similar to those of the control mice.3. The cell proliferation in Twist1and Twist2double heterozygous mice were reduced.We noticed that the BrdU-positive cells in the area of the metaphysis, as well as in the mid diaphyseal periosteum and cortical bone, were significantly reduced in the Twist1flox; Twist2Cre/+mice. However, no difference in the osteoblast apoptosis was observed.4. FGF signaling pathway was downregulated in bone and tooth in Twistl and Twist2deficient mice.Quantitative real-time PCR revealed a substantial decrease in the transcript levels of Fg/2and Fgfrs1,2,3and4in the Twist1flox/+; Twist2Cre/+mice. Immunohistochemistry further confirmed that the Fgfr2protein was markedly reduced in the long bones of the Twist1folx/+; Twist2Cre/+mice. Consistent with the reduced Fgf2and Fgfr expression, the immunohistochemistry revealed that the levels of phospho-Erkl/2were considerably lower in Twist1flox/+; Twist2Cre/+mice than in the control mice. Accordingly, quantitative real-time PCR demonstrated that the levels of Erm and Pea3transcripts were significantly downregulated. Tooth-related FGF genes including Fgf3, Fgf10, Fgfrl, and Fgfr2were also dected by quantitative real-time PCR by using dissected tooth bud samples of Twist1flox/flox, Twistcre/+mice and control mice at E14.5. Similarly, these genes showed decreased expression level.5. Twist1/2and E12synergistically stimulate the4.9kb Fgfr2promoter activities. Our promoter luciferase reporter assay revealed that Twistl or Twist2alone was able to stimulate a4.9kb Fgfr2promoter fragment in the odontoblast-like MDPC-23cells but not in the C3H10T1/2mesenchymal cells or MC3T3-E1pre-osteoblast cells. However, they strongly enhanced the stimulatory activity of E12. a ubiquitously expressed Twist binding partner, in all the above cell lines.Conclusions:1. Twistl and Twist2have synergistical and positive role in regulating bone formation.2. Twistl and Twist2regulate bone development by affecting osteoblast differentiation and proliferation.3. FGF signaling pathway is compromised in Twistl and Twist2double heterozygous mice.4. Twistl and Twist2regulate cusp formation by modulating FGF signaling5. Twistl/2and E12form heterodimers and positively regulate Fgfr2promoter activity. Introduction:Cleidocranial dysplasia (CCD) is a skeletal disorder with autosomal dominant inheritance caused by haplo-insufficiency of RUNX2gene. The disease is characterized by dental abnormalities, incomplete or late closure of cranial sutures, rudimentary or absent clavicles and other skeletal anomalies.Subjects and Methods:The phenotypes of7patients from two unrelated CCD families and1patient with unproven family history were investigated and the subcellμlar localization of the mutant RUNX2was assessed.Results:Three RUNX2frameshift mutations were identified. Two of the mutations are novel (c.887insC and c.592delA) and one (c.90insC) has been described previously. Subcellμlar localization analysis showed that c.592delA and c.90insC, both of which produced a stop codon in the Runt domain, caused only a partial loss of nuclear translocation, whereas the wild type and c.887insC RUNX2proteins were localized exclusively in the nucleus.Conclusions:Our findings support the notion that haplo-insufficiency of RUNX2is responsible for CCD; in addition we show that the absence of the best characterized nuclear localization signal (NLS) does not totally abolish the nuclear translocation of truncated RUNX2suggesting that RUNX2contains a second nuclear localization signal within or at the N-terminal border of the Runt domain.