Morphological study of tooth development in podoplanin-deficient mice

Podoplanin is a mucin-type highly O-glycosylated glycoprotein identified in several somatyic cells: podocytes, alveolar epithelial cells, lymphatic endothelial cells, lymph node stromal fibroblastic reticular cells, osteocytes, odontoblasts, mesothelial cells, glia cells, and others. It has been reported that podoplanin-RhoA interaction induces cytoskeleton relaxation and cell process stretching in fibroblastic cells and osteocytes, and that podoplanin plays a critical role in type I alveolar cell differentiation. It appears that podoplanin plays a number of different roles in contributing to cell functioning and growth by signaling. However, little is known about the functions of podoplanin in the somatic cells of the adult organism because an absence of podoplanin is lethal at birth by the respiratory failure. In this report, we investigated the tooth germ development in podoplanin-knockout mice, and the dentin formation in podoplanin-conditional knockout mice having neural crest-derived cells with deficiency in podoplanin by the Wnt1 promoter and enhancer-driven Cre recombinase: Wnt1-Cre;PdpnΔ/Δmice. In the Wnt1-Cre;PdpnΔ/Δmice, the tooth and alveolar bone showed no morphological abnormalities and grow normally, indicating that podoplanin is not critical in the development of the tooth and bone.


Introduction
Podoplanin is a mucin-type highly O-glycosylated [1] glycoprotein identified in the rat kidney glomerular epithelial cell (podocyte) foot processes [2]. Podoplanin is highly negative charged by a high sialic acid content [1][2][3][4][5][6]. Mouse 38-kDa and human 36-kDa podoplanin cloned from lymphoid tissue have been termed gp38 [7] and gp36 [8] respectively. Podoplanin is commonly known as a useful lymphatic endothelial cell marker because blood endothelial cells lack podoplanin. Podoplanin null mice have defects in the lymphatic pattern formation with pronounced lymphedema at birth and show blood-filled lymphatics [9][10][11]. Kato et al. have reported details of podoplanin bindings to the platelet transmembrane protein CLEC-2 causing platelet agglutination by the binding activity [6]. It has been reported that the platelet a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 agglutination on the lymphatic endothelium plays a key role in the sprouting of two jugular lymph sacs from the junction of the subclavian veins with the anterior cardinals (future internal jugular vein) [11]. Further, it has been established that podoplanin interact with Rho GTPase for cell process stretching [12]. Podoplanin plays a key role in the lymph node expansion by signaling between lymph node stromal fibroblastic reticular cells and CLEC-2 delivering dendritic cells [13]. The CLEC-2 binding rapidly causes podoplanin uncoupling from RhoA and permits cytoskeleton relaxation in reticular cells, and cell process stretching [13].
Overall, this suggests that podoplanin plays a number of different roles in contributing to cell functioning and growth by signaling. It appears likely that podoplanin plays a critical role in the development of the type I alveolar lung cells, and in the lymphatic circulation system and lymph node expansion in vivo, and that podoplanin-RhoA interaction induces cytoskeleton relaxation and the cell process stretching in fibroblastic cells, osteocytes, and other cell lines in vitro. However, the function of podoplanin in other podoplanin-positive organs, particularly in the hard tissue, has not been established as podoplanin absence is lethal to mice because of the occurrence of respiratory failure at birth as mice lines with podoplanin-floxed alleles have not yet. In Wnt1-Cre transgenic mice which express Cre recombinase under the control of the wingless-related MMTV integration site 1 (Wnt1) promoter and enhancer have been extensively used in the study of the neural crest derivatives [41]. Here, we report the tooth and alveolar bone development in podoplanin-conditional knockout mice in which podoplanin is absent in neural crest-derived cells: Wnt1-cre transgenic mice bred to mice having podoplanin-floxed alleles (Pdpn fl/fl ).

Animals
The experimental protocol for animal use was reviewed and approved by the Animal Experiment Committee of Fukuoka Dental College in accordance with the principles of the Helsinki Declaration. Breeding and experiments were performed in a room with a 100% controlled atmosphere which had passed an examination for bacteria and is located in the Fukuoka Dental College Animal Center. Mice grew normally and lived healthily under conventional atmosphere conditions with normal feeding in cages and rooms in which the temperature (22˚C) and humidity (55%) were completely controlled. The mice were housed with an inverse 12 hour day-night cycle with lights on from 7:00pm.
Humane endpoints were used in the experiments as a rapid and accurate method for assessing the health status of the mice, that is, mice with lost ability to ambulate (inability to access food or water) were euthanized by induction anesthesia (1 l/min of 2% isoflurane mixed with 30% oxygen and 70% nitrous oxide with an anaesthetic apparatus) followed by cervical dislocation and intraperitoneal injections with 3.5% chloral hydrate (10 ml/kg, trichloroacetaldehyde monohydrate, Kanto Chemical, Tokyo, Japan) in the saline.

Generation of knockout first
The targeting vector of the podoplanin gene (Pdpn) was purchased from EUCOMM (European Conditional Mouse Mutagenesis Program) which allows reporter-tagging and conditional mutation of the gene-of-interest and the generation of knockout first was entrusted to Transgenic Inc. (Fukuoka, Japan): allele name, Pdpn tm1a(EUCOMM)Wtsi ; genetic background, C57BL/6N-A tm1Brd (Fig 1A). The targeting vector is the promoter-driven targeting cassette and consists of the gene-trap cassette followed by the selection cassette through the loxP site. The targeting vector has three loxP sites with the first and second loxP sites sandwiching the gene-trap cassette. The gene-trap cassette contains a splice acceptor (SA) and an internal ribosome entry site (IRES) upstream of a lacZ reporter gene followed by a polyadenylation (pA) signal. The IRES-lacZ trapping cassette is placed at 5 0 of a loxP-flanked, promoter-driven, neomycin-resistance selection cassette, which lies immediately upstream of exon 3. The lacZ cassette is able to apply in the generation of reporter-tagged animals expressing lacZ in the tissue that expresses the gene-of-interest. The selection cassette consists of a neomycin resistance gene (NeoR) driven by an autonomous promoter (hBactP) and pA signal. When the targeting vector, HTGR03003_Z_2_G05, is successfully inserted in the Pdpn gene domain downstream of the promoter, the gene is inactivated, and LacZ expresses instead. At EUCOMM this genetrap cassette is used as a consensus structure of the targeting vector. The targeted allele Pdpn gt , containing the gene trap and selection cassettes is a conventional knockout-first allele [42], as the insertion of the cassettes disrupts the targeted gene splicing in C57BL/6N embryonic stem (ES) cells [42]. The Pdpn gene knockout-first allele, also referred to as Pdpn KO1st , was made by the reporter-tagged insertion with conditional potential (Transgenic Inc., Fukuoka, Japan). The Pdpn KO1st mice were generated from chimeric mice with the Pdpn-targeted ES cells in which the genetic background is C57BL/6NCrj. Although the cassettes should be removed before the investigation of the phenotype development in the Pdpn KO1st , it is thought that Pdpn KO1st mice containing Pdpn gt homozygously are useful because of the absence of anomalies in mice having Pdpn gt heterozygously, and the insertion of the cassettes would not affect the gene around the Pdpn gt allele in this case. The promoter-driven targeting cassette which consists of the gene-trap and selection cassettes is flanked by flippase (Flp) recognition target FRT sites; one the upstream of the IRES-lacZ cassette and one between the upstream of the neomycin-resistance cassette and the second loxP site before exon 3 in order to simultaneously remove both the gene trap IRES-lacZ and the neomycin-resistance cassettes. The removal of . The cassette is composed of a short flippase recombination enzyme (Flp)-recognition target (FRT), reporter, and a Cre recombinase recognition target (loxP). Cre-loxP system from bacteriophage P1 is analogous to Flp-FRT system from Saccharomyces cerevisiae and recombines a pair of target sequences. The first FRT site is followed by the reporter which is a reading frame-independent LacZ gene trap cassette: splice acceptor of mouse En2 exon 2 (En2-SA), the internal ribosome entry site from encephalomyocarditis virus (ECMV IRES), Escherichia coli lacZ gene encoding the reporter enzyme β-galactosidase (lacZ), and simian virus 40 polyadenylation signal (pA). The first loxP site is followed by the neomycin selection cassette which is composed of human beta-actin promoter (hBactP) driving the neomycin-resistance gene (selectable marker neomycin phosphotransferase, neo), pA, a second FRT site, and a second loxP site. A third loxP site is inserted downstream of the targeted exon (synthetic loxP region, 22973-23052) at position 143273615, therefore, Pdpn exon 3 is flanked by loxP sites. A reporter knockout mouse not crossed with flp deleter mouse was used as Pdpn KO 1st mouse. Breeding with flp recombinase-deleter mouse created mouse carrying a floxed allele by subsequent excision of the targeting cassette. This study did not use mice removed the βact-neo cassette and the critical exons, and carrying a lacZ tagged null allele by applying Cre recombinase to the original version of the allele. Subsequently breeding Pdpn-floxed mouse with Wnt1-Cre mouse created mouse carrying a Pdpn exon3 null allele in the Wnt1-expressng cells. Cre-Lox analogous to Flp-FRT recombination is a site-specific recombination system consists of a Cre recombinase that recombines a pair of short target sequences called the lox sequences. The Cre enzyme and the original lox site called the loxP sequence are derived from bacteriophage P1. Tooth development in podoplanin-deficient mice the targeting cassette by Flp generates a Pdpn conditional KO allele including loxP sites flanking exon 3 (floxed exon 3). The exon3 is common to all Pdpn transcript variants and is critical in podoplanin protein development. The exon3 deletion creates a frame-shift mutation in the Pdpn expression. A third loxP site is inserted immediately after the exon 3 to remove the exon 3 and generate the Pdpn-inactivated mice in which both alleles of Pdpn exon 3 are homozygous null. Mice carrying the Pdpn gt allele heterozygously were first bred with mice carrying a ubiquitously expressed Flp, ACTB:FLPe (B6;SJL-Tg(ACTFLPe)9205Dym/J, JAX 003800), in order to remove the promoter-driven targeting cassette by the Flp-mediated recombination in vivo. This breeding left a single FRT site and floxed exon 3 behind, enabling a true conditional knock-out by the deletion of exon 3. This allele is referred to as Pdpn fl . Mice carrying both the homozygous Pdpn fl alleles (Pdpn fl/fl ) and the Wnt1-expressing tissue specific heterozygous Cre recombinase gene were generated to obtain mice for the Wnt1-expressing tissue specific deletion of Pdpn exon 3, using Pdpn fl/fl mice and the Cre recombinase gene transgenic mice: B6. Cg-Tg(Wnt1-Cre)11Rth Tg(Wnt1-GAL4)11Rth/J (C57BL/6JxCBA/J:Wnt1-Cre, Jax 009107). Deletion of exon 3 causes a frame shift and starts a premature stop codon near the 5 0 end of exon 4 or 5 depending on splicing variants, thereby disrupting translation of the Pdpn. This allele is referred to as Pdpn Δ . Deficiency of podoplanin by the Wnt1 promoter and enhancerdriven Cre recombinase occurs in the neutral crest-derived tissue expressing Wnt1-Cre in the Wnt1-Cre;Pdpn Δ/Δ mice. We examined the conditional mutant C57BL/6NJxCBA/J:Wnt 1-Cre; Pdpn Δ/Δ mice with the Wnt1-expressing tissue specific Pdpn deletion (Wnt1-Cre;Pdpn Δ/Δ ), as well as the wild-type (Pdpn +/+ ) and heterozygous (Pdpn Δ/+ ) littermate controls carrying the Cre transgene.

Genotyping
Genomic DNA from tail was isolated with a QIAamp DNA Blood and Tissue Kit (Qiagen, Hilden, Germany). All procedures were performed according to protocols provided by the manufacturers, and, in all cases, the duration of each procedure was recorded. The PCR was performed by 30 cycles for amplification using the Ex Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50 pM of primer sets: The Pdpn targeted allele with the whole trapping cassette (6.5kbp); Pdpn_sc5AF1 (forward) 5'-CAGTGAGATTCTATAGGGCTGC, LacZ_AS1 (reverse): 5'-TTGTAAAACGACGGGATCTTCC; Pdpn without loxP site (wild, 133bp) and Pdpn including the third loxP site (208bp)(synthetic loxP region, 22973-23052)( Fig 1B); loxS1 (forward) 5'-AGGAAGAATCCCACACCAGG, loxAS1 (reverse): 5'-TGTAGGGAGCTACCGC TAGG. The primer sets of loxS1 and loxAS1 were basically used for detecting mice having Pdpn KO1st and Pdpn fl/fl alleles ( Fig 1B). The PCR products were separated on 2% agarose gel (NuSieve; FMC, Rockland, ME, USA) and visualized by Syber Green (Takara). The correct size of the amplified PCR products was confirmed by gel electrophoresis and the amplification of accurate targets was confirmed by sequence analysis. The Cre recombinase gene driven by Wnt1 promoter/enhancer (KC845567) was detected by PCR products (472bp) (Fig 1C) using the primer sets: 5'-CGTTTTCTGAGCATACCTGGA (forward), 5'-ATTCTCCCACCGTCAG TACG (reverse).

Immunohistochemistry
Kawamoto's film method with tungsten carbide blade was used for the sectioning so that intact hard and soft tissue can be observed without decalcification [43]. After the subjects were embedded in super cryoembedding medium (Leica Microsystems Japan, Tokyo, Japan) and rapidly frozen using liquid N 2 , sagittal undecalcified frozen sections (4 μm) of tissue including the upper incisor region were cut in a cryostat (Leica Microsystems, Wetzlar, Germany) with tungsten carbide blade. The sections were fixed in 100% ethanol for 30 sec at RT and subsequently immersed in 100% methanol for 30 sec at -20˚C, treated with 0.1% goat serum for 30 min at 20˚C, and then treated for 8 hrs at 4˚C with PBS containing 0.1% goat serum and the following primary antibodies (1 μg/ml): hamster monoclonal anti-mouse podoplanin (Angio-Bio Co., Del Mar, CA) and rabbit anti-nephrin (Abcam plc., Cambridge, UK). After the treatment with primary antibodies the sections were washed three times in PBS for 10 min and immunostained for 0.5 hr at 20˚C with 0.1 μg/ml of the second antibodies: Alexa Fluor (AF) 488 or 568-conjugated goat anti-hamster IgG and anti-rabbit IgG (Probes Invitrogen Com., Eugene, OR). The immunostained sections were mounted in 50% polyvinylpyrrolidone solution and examined by fluorescence microscopy (BZ-8100, Keyence Corp., Osaka, Japan) or confocal laser-scanning microscopy (LSM710, Carl Zeiss, Jena, Germany) with an x63 oil Plan Apochromatic objective lens.

Measurement of the area of immunostaining
The podoplanin-stained area was measured on the five different spots (0.36 mm x 0.36 mm) in the renal section images using ImageJ (National Institutes of Health, Bethesda, MD). The relative expression amounts of podoplanin were expressed by the mean of the ratio: podoplaninpositive area in lung lobes (x20 magnification) and alveoli (x200 magnification) / scanned area.

Statistics
All experiments were carried out five times, repeatedly, and data are expressed as mean + SD. The statistical significant differences (p< 0.01) were determined by one-way ANOVA and the unpaired two-tailed Student's t test with STATVIEW 4.51 software (Abacus concepts, Calabasas, CA, USA).

Immunostaining of Pdpn KO1st mice for podoplanin
In the kidney of Pdpn KO1st mice, the expression of podoplanin was observed in the glomeruli of Pdpn +/mice as well as the wild type Pdpn +/+ mice, but not in the Pdpn -/mice (Fig 2). The expression of nephrin was observed in Pdpn -/and Pdpn +/mice, as well as in the wild type mice. It was observed that the podocytes express podoplanin and nephrin, and the area of the diaphragm between podocytes express only nephrin. There were no abnormalities in the development of glomeruli in the Pdpn +/and Pdpn -/mice.
In the thoracic cage and lung of Pdpn KO1st mice, there were no abnormalities in the development of pleura and costal bone in the Pdpn +/and Pdpn -/mice but atrophy of the lung was observed in the Pdpn -/mice (Fig 3). In the Pdpn +/+ and Pdpn +/mice, it was observed that the terminal ends of the respiratory tree, the pulmonary alveoli, consist of alveolar sacs and alveolar ducts whereas there were fewer developed intact alveoli in the Pdpn +/mice than in the Pdpn +/+ mice, and there was disordered development in the Pdpn -/mice (S1 Fig). The expression of podoplanin was observed in the pleura mesothelial cells, costal bone, and lung parenchyma of Pdpn +/mice as well as in the Pdpn +/+ mice, but not observed in the Pdpn -/mice. The podoplanin expression of the alveoli in the Pdpn +/mice was weaker than in the Pdpn +/+ mice, but not observed in the Pdpn -/mice. In ImageJ analysis for the immunostained area, the podoplanin expression amounts on lung lobes and alveoli diaphragmatic pleura are significantly higher in the wild type Pdpn +/+ mice than in the Pdpn +/mice (S2 Fig). In the Pdpn +/+ and Pdpn +/mice, there were podoplanin-positive type I alveolar epithelial cells among the thyroid transcription factor-1 (TTF-1)-positive type II alveolar epithelial cells (Fig 4). The podoplanin-positive area of the type I alveolar epithelial cells in Pdpn +/- mice were smaller than in the wild type Pdpn +/+ mice. In the Pdpn -/mice, the terminal ends of the respiratory tree consisted of the alveolar duct and respiratory bronchioles with only TTF-1-positive type II alveolar epithelial cells, but lacked alveolar sacs with podoplanin-positive type I alveolar epithelial cells.
In the mandible of wild type mice, the expression of podoplanin was observed in the tooth germ, nerve sheaths, and Meckel's cartilage (Fig 5). In the tooth germ of the wild type mice, the expression of podoplanin was observed in the enamel cord, cervical loop, inner and outer enamel epithelia, and odontoblasts. In the mandible of Pdpn KO1st mice, the expression of podoplanin was observed in the tooth germ, nerve sheaths, and alveolar bone in the Pdpn +/mice, whereas it was not observed in the Pdpn -/mice (Fig 6). The expression of podoplanin in the mandible was weaker in the Pdpn +/mice than in Pdpn +/+ mice. There seems to be no In the hematoxylin-eosin (HE) staining, atrophy is observed in the Pdpn -/mice. The expression of podoplanin (arrowheads) is observed in the lung of the wild type Pdpn +/+ mice and Pdpn +/mice, but not in the Pdpn -/-. It is observed that the PDPN-positive areas are the mesothelia (arrowheads) of diaphragmatic pleura, costal pleura, and visceral pleura. It is also observed that costal bone (arrows) and lung parenchyma (asterisks) are PDPN positive. Staining densities of parenchyma PDPN are weaker in the Pdpn +/mice than in the Pdpn +/+ mice. Bar: 1 mm. abnormalities in the development of tooth germs, bone, or nerves in the Pdpn +/and or Pdpn -/mice apparently (Fig 6).

Immunostaining of Wnt1-Cre;Pdpn conditional knockout mouse tooth for podoplanin
In the 2-week Wnt1-Cre;Pdpn Δ/+ mouse incisors, the expression of podoplanin was observed in the Hertwig's epithelial root sheath, odontoblasts, apical bud, inner and outer enamel epithelial cells and pre-ameloblasts (Figs 7 and 8). The podoplanin expression was weaker in the odontoblasts where dentin formation progressed (Fig 9) and in the ameloblasts where the enamel formation had started (Fig 10). The podoplanin expression was also observed in the periosteum at the edge of the alveolar bone and the nerve sheath while it was not observed in the dentin, dental pulp fibroblasts, pre-odontoblasts, periodontal ligament, bone marrow, or muscle. There were no abnormalities in the nerve, bone, or tooth development.
In the 2-week Wnt1-Cre;Pdpn Δ/Δ mouse lower incisor, the expression of podoplanin was observed in the epithelial cells: Hertwig's epithelial root sheath, apical bud, inner and outer enamel epithelial cells, and pre-ameloblasts, whereas not observed in the odontoblasts, bone, or nerve sheath, which were PDPN-positive in the Pdpn fl/fl (not shown) or Wnt1-Cre;Pdpn Δ/+ mice (Figs 11-14). The expression of PDPN in the ameloblasts where enamel formation had started was weaker than in pre-ameloblasts without enamel formation (Fig 14). There were no abnormalities in the nerve, bone, or tooth development (Figs 11-14). Tooth development in podoplanin-deficient mice

Discussion
Podocyte development in the podoplanin-deficient kidney in the Pdpn KO1st mice Podoplanin has been identified in rat kidney podocyte foot processes [2]. Nephrin is a transmembrane protein that is a structural component of the slit diaphragm. The two are present on the podocytes and maintain the relationship between the basement membrane and podocytes. Nephrin is specifically located at the slit diaphragm of glomerular podocytes. In this study, the expression of podoplanin on podocytes were observed in Pdpn +/mice as well as in the wild type (Fig 2). The expression of nephrin was observed on podocytes and in the area of the diaphragm between podocytes in the wild type, Pdpn +/-, and Pdpn -/mice, and there were no anomalies in the development of glomeruli in the Pdpn -/mice, suggesting an absence of podoplanin in podocytes causes no morphological disorder in the podocyte processes.

Fig 6. Immunostaining of a Pdpn KO1st mouse lower incisor tooth germ sagittal section at the late bell stage for podoplanin (PDPN).
In the hematoxylin-eosin (HE) staining, there are no abnormalities in the tooth germ with dentin matrix, bone, or nerve of the Pdpn +/and Pdpn -/mice. In the left immunostained images, the expression of PDPN is observed in the tooth germ (asterisks), in the alveolar bone (arrows), and in the sheath of the inferior alveolar nerve (arrowheads) of the wild type Pdpn +/+ mice, and in the Pdpn +/mice to a weaker extent than in the Pdpn +/+ mice. There is no immunostaining observed in the Pdpn -/tissue except for a non-specific reaction. In the right images at the higher magnification of the parts highlighted by the boxes in the left images, the expression of PDPN is observed in the odontoblasts (arrowheads), and in the inner enamel epithelial cells (arrows), of the wild type Pdpn +/+ mice, and of the Pdpn +/mice to a weaker extent than in the Pdpn +/+ mice. There is no immunostaining observed in the Pdpn -/tissue except for the cross reaction to the dentin matrix. Bar: 100μm.  Type I alveolar lung cells express podoplanin [19,20], and the extremely flat and thin type I alveolar cells function as an air-blood barrier. It has been thought that the type II alveolar lung cells differentiate into type I lung cells by podoplanin signaling, and podoplanin expression has been reported in osteocytes and osteoblasts [14][15][16]. In culture, the podoplanin production is more active, and the amounts are larger in MLO-Y4 osteocyte-like cells than in osteoblasts. Mechanical stress application increases the production of podoplanin and increases the number and lengths of dendrites of the osteocyte-like MLO-Y4 cells. This has led to the hypothesis that podoplanin controls osteocyte morphology and is essential for normal bone function in Tooth development in podoplanin-deficient mice response to shear stresses. Podoplanin expression has also been reported in mesothelial cells [24,25]. In the thoracic cage and lung in this study, the expression of podoplanin was observed in the pleura mesothelial cells, costal bone, and lung parenchyma in the wild type mice and in the Pdpn +/mice, but not in the Pdpn -/mice (Fig 3). The PDPN expression of the alveoli in the Pdpn +/mice was weaker than in the wild type mice. There were no abnormalities in the development of pleura and costal bone in the Pdpn +/and Pdpn -/mice, but there was atrophy of the lung and disorder of the alveoli observed in the Pdpn -/mice (Fig 3). ImageJ analysis for the immunostaining of lung showed that the expression amounts of podoplanin on lobes and alveoli were significantly higher in the wild type Pdpn +/+ mice than in the Pdpn +/mice (S1 and S2 Figs). There may be difference between allele products for podoplanin. In the wild type and the Pdpn +/mice, there were PDPN-positive type I alveolar epithelial cells among the TTF-1-positive type II alveolar epithelial cells (Fig 4). The PDPN-positive type I alveolar epithelial cells were fewer in Pdpn +/mice than in the wild type Pdpn +/+ mice. In the Pdpn -/mice, the alveoli consisted of the alveolar duct with TTF-1-positive type II alveolar epithelial cells but lacked alveolar sacs with type I alveolar epithelial cells. It may be suggested that type II alveolar lung cells with TTF-1 fail to differentiate into type I lung cells in the absence of podoplanin, and that the podoplanin deficient causes no morphological disorder in the development of the pleura and costal bone. Podoplanin expression has been reported in enamel epithelial cells and odontoblasts in the tooth germ [27,29]. In this study, the expression of podoplanin was observed in the tooth germ, craniofacial bone, nerve sheaths, and Meckel's cartilage in the wild type mice (Fig 5). In Tooth development in podoplanin-deficient mice the tooth germ, podoplanin expression was observed in the enamel cord, cervical loop, inner and outer enamel epithelia, and odontoblasts (Fig 5). The PDPN expression in the tooth germ and craniofacial bone was also observed in the Pdpn +/mice to a lesser extent than the wild type mice, but it was not in the Pdpn -/mice (Fig 6). Further, there were no abnormalities in the development of tooth germs, craniofacial bone, and nerves in the Pdpn +/and Pdpn -/mice, suggesting that the podoplanin deficient causes no morphological disorder in the development of tooth germ and craniofacial bone. The Wnt1 plays an important role in anterior-posterior patterning in the embryonic central nervous system, and Wnt1 is also expressed in cranial neural crest-derived craniofacial and odontogenic mesenchymal cells differentiating into odontoblasts, chondrocytes, and osteoblasts [41,[44][45][46]. Odontogenesis starts with dentinogenesis by the secretion of predentin from odontoblasts following the terminal differentiation controlled by the inner enamel epithelium. Subsequently, the inner enamel epithelium differentiates into ameloblasts by dentin matrix interactions, and ameloblasts secrete enamel matrix. Podoplanin is not expressed in dental pulp cells and pre-odontoblasts, but is expressed in odontoblasts during dentinogenesis, and the podoplanin expression in odontoblasts diminished after dentin formation. Podoplanin Tooth development in podoplanin-deficient mice is also expressed in the inner and outer enamel epithelical cells, and is lost in the ameloblasts starting the enamel matrix secretion [27,29]. In the 2-week Wnt1-Cre;Pdpn Δ/+ mouse incisors, the expression of PDPN was observed in the oral epithelial cells and oral epithelial cell-derived tooth germ epithelial cells: Hertwig's epithelial root sheath, apical bud, and inner and outer enamel epithelial cells, and in the periosteum at the edge of the alveolar bone and the nerve sheath, as well as in the wild type mice and Pdpn fl/fl (not shown) (Figs 7 and 8). The expression of podoplanin is not observed in the dental pulp cells or in pre-odontoblasts, but it was In the hematoxylin-eosin (HE) staining, there are no abnormalities in the dentin and enamel formation. In the left images at the higher magnification of the parts highlighted by the box (d) in Fig 11, PDPN expression is not observed in the odontoblasts (Ob). The immunoreaction is not in the dentin (De) but cross-reaction is observed in the enamel (En). In the right images at a higher magnification of the parts highlighted by box in the left images, podoplanin expression is observed in the outer enamel epithelial cells (OEE), and in the ameloblasts (Ab) with enamel formation to a smaller extent than in the inner enamel epithelial cells without enamel formation before differentiation into the ameloblasts. Bar: left 100μm, right 10μm. Tooth development in podoplanin-deficient mice observed in odontoblasts, and to a smaller extent in the odontoblasts where dentin formation had progressed and this was also the case in the wild type mice (Fig 9). The PDPN expressions was also observed in pre-ameloblasts, but less so in the ameloblasts where enamel formation had started, as well as in the wild type mice and Pdpn fl/fl , corresponding to the podoplanin expression pattern previously reported (Fig 10) [27,29]. Therefore, it may be assumed that the insertion of a targeting vector and the deletion of the promoter-driven targeting cassette, remaining two loxP sites before and after Pdpn exon3, did not affect to the podoplanin production. There was no cross-reaction to the connective tissue and muscle, suggesting that the immunostaining is successful.
In the 2-week Wnt1-Cre;Pdpn Δ/Δ mice, there was no expression in the odontoblasts, in the osteocytes of the alveolar bone, or mandibular nerve sheaths, which were PDPN-positive in the Wnt1-Cre;Pdpn Δ/+ mice, whereas the expression of PDPN was observed in the oral epithelial cells and oral epithelial cell-derived tooth germ epithelial cells: Hertwig's epithelial root sheath, apical buds, and inner and outer enamel epithelial cells (Figs 11-14). The neural crestderived odontoblasts, chondrocytes, and osteoblasts express Wnt1 [41,[44][45][46]. Therefore, it may be assumed that the productions of Pdpn fl/fl and podoplanin-conditional knockout in the Wnt1 expressing cells were successful. It has been established that podoplanin plays a key role in the elongation of cell processes by the signaling with RhoA family proteins [12,13]. Since podoplanin expression is dependent on odontoblast differentiation, and is reduced in the odontoblasts and ameloblasts as the dentin and enamel formation progresses, we expected that podoplanin could be a contributing factor in the elongation of odontoblast cell processes and dentin formation. However, there were no morphological anomalies in the alveolar bone or tooth in the 2-week Wnt1-Cre;Pdpn Δ/Δ mice with podoplanin-deficient odontoblasts and osteocytes (Figs 11-14). The expression of podoplanin in the ameloblasts which had started enamel formation was weaker than in the pre-ameloblasts where no enamel formation had taken place, as was the case in the wild type and Wnt1-Cre;Pdpn Δ/+ mice (Fig 14). In the Wnt1-Cre; Pdpn Δ/Δ adult mice, the tooth and alveolar bone has grown normally. Therefore, it may be suggested that podoplanin expression in odontoblasts is not associated with the dentin and enamel formation, and that podoplanin expression in osteoblasts does not play a critical role in the bone maintenance under usual circumstances without any stress.
Supporting information S1 Fig. Immunostaining of the respiratory tree terminal ends in the Pdpn KO1st mice for podoplanin (PDPN). In the hematoxylin-eosin (HE) staining, the development of intact alveoli (asterisks) is more frequent in the Pdpn +/+ mice than in the Pdpn +/mice, whereas alveolar sacs are disordered in the Pdpn -/mice. The expressions of podoplanin on the alveoli (arrowheads) and on the mesothelia of diaphragmatic pleura (arrows) are observed in the wild type Pdpn +/+ mice and the Pdpn +/mice, but not in the Pdpn -/mice. In the Pdpn +/+ and Pdpn +/mice, the terminal ends of the respiratory tree, pulmonary alveoli, are found in the lung parenchyma and consists of alveolar sacs and alveolar ducts. The PDPN expression of alveoli in the Pdpn +/mice is weaker than in the Pdpn +/+ mice. Bar: 100μm. (TIF) The relative expression amounts of podoplanin were expressed by the mean of the ratio (%): podoplanin-positive area in lung lobes (x20, Fig 3) and alveoli (x200, Fig 4) / scanned area. The expression amounts of podoplanin on lung lobes are significantly higher in the wild type Pdpn +/+ mice than in the Pdpn +/mice. Ã Significant in ANOVA (P<0.001). (TIF)