BACKGROUND
development of the dentition is regulated by time- and position-specific reciprocal epithelial-mesenchymal interactions .
these odontogenic interactions are directed and coordinated by transcription factors, growth and signaling factors, their cognate receptors, and extracellular matrix constituents  <cit> .
collectively, various combinations of molecules within these interactions determine when or where teeth develop and modulate the specifications for tooth size and shape.
noggin, wnt5a, and mfrzb <dig> genes have been shown to regulate tooth size .
cai et al., using hetero-specific tissue recombination from rat molar tooth organs, demonstrated that tooth size is determined not by dental papilla mesenchymal cell number, but by intrinsic tissue-specific dental papilla mesenchymal factors  <cit> .
in addition, exogenous treatment with bone morphogenic protein   <dig>  noggin, fibroblast growth factor   <dig>  and fgf <dig> does not affect tooth size, despite the observation that bmp2/ <dig>  fgf3/ <dig>  lef <dig>  and wnt5a/5b are expressed in the dental mesenchyme during the cap and early bell stages of tooth morphogenesis .
the crown width of a bioengineered molar reconstructed with dissociated epithelial and mesenchymal cells through the organ germ method is correlated with the length of the contact area between the epithelial and mesenchymal cell layers  <cit> .
in conditional bmp7-deficient embryos, the maxillary incisor tooth organs are either missing or hypoplastic, and the development of the first molar tooth organs is delayed, malformed, or missing  <cit> .
therefore, bmp <dig> is assumed to be an essential growth factor for tooth morphogenesis.
bmp <dig> is a 35-kda homodimeric protein that is associated with a variety of signaling pathways, including the canonical smad pathway, mitogen-activated protein kinase -related pathways, and the phosphoinositol 3-kinase /akt pathway .
uterine sensitization-associated gene- <dig>  is a heavily glycosylated 28–30 kda secretory protein that functions as a monomer to induce signal transduction.
usag- <dig> binds to the wnt coreceptors low-density lipoprotein receptor-related protein   <dig> and lrp <dig> and inhibits wnt signaling  <cit> .
lrp <dig> modulates and integrates bmp and canonical wnt signaling during tooth morphogenesis by binding to secreted usag- <dig>  <cit> .
usag- <dig> expression is further restricted to the distal renal tubules, in a pattern similar to the localization of bmp <dig>  usag- <dig> is a bmp antagonist; it interacts with bmp <dig> in the developing and adult kidney and directly binds to bmp2/4/ <dig>  as assayed using co-immunoprecipitation studies  <cit> .
moreover, we previously reported that usag-1-deficient mice exhibit supernumerary maxillary incisors in response to enhanced bmp signaling and that bmp signaling is modulated by wnt signaling in usag-1-deficient mice  <cit> .
based on these observations and findings, we hypothesized that bmp <dig> and usag- <dig> play important roles in tooth morphogenesis.
therefore, we previously established double genetically modified mice to analyze the in vivo inter-relationships between bmp <dig> and usag- <dig>  using this model, we demonstrated that usag- <dig> inhibits bmp <dig> signaling, leading to apoptosis and degeneration of rudimentary tooth germs in maxillary supernumerary incisor formation  <cit> .
moreover, we found that the tooth size in mandibular incisors of bmp <dig> heterozygous mice was altered.
accordingly, in this study, we examined the effects of bmp <dig> and usag- <dig> signaling on tooth size in mandibular incisors using the f <dig> generation of mice.
methods
production and analysis of usag-1- and bmp7-lacz knock-in mice
bmp7-lacz knock-in  mice and usag1-lacz knock-in  mice were produced as previously described  <cit> .
bmp7-deficient mice were embryonic lethal.
day e <dig> was established as midnight prior to finding a vaginal plug.
polymerase chain reaction  amplification was performed using kod fx neo polymerase  and specific primers for genotyping.
x-gal staining
the bacterial lacz  gene in bmp7lacz/lacz  and usag1lacz/lacz  mice was knocked into the gene of interest  <cit> .
frozen sections of embryos were fixed in 4 % paraformaldehyde for 2 min and stained with x-gal  twice as whole mounts and frozen sections, followed by counterstaining with nuclear fast red .
for x-gal staining, embryos were incubated at 37 °c in buffer  <dig>  35 mm k4fe <dig>  2 mm mgcl <dig>   <dig>  % np- <dig>   <dig>  % sodium deoxycholate, 1× phosphate-buffered saline ).
we observed mandibular incisors in bmp7+/−  and usag-1+/−  mice and mandibular molars in usag-1+/−  mice at e <dig> and e <dig>
analysis of the adult tooth phenotype
three-dimensional  computed tomography  scans  were performed using the heads of adult mice.
we converted cb files to tiff files.
cb files had 512 × 512 pixels,  <dig> bits, and a voxel size of x:y:z = 1:1: <dig> .
next, 3d images were reconstructed and analyzed with computer imaging software   <cit> .
the mandibular incisor volume was measured using the imaging software; then, to measure the cross-sectional area, mandibular incisors were cut vertically to the tooth axes at the uppermost point on the incisor alveolar rim , so as to avoid the influence of environmental factors, such as dental occlusion and tooth attrition.
the cross-sectional area of the incisors was measured using imagej software.
we analyzed a total of  <dig> wild-type  and  <dig> bmp7+/− samples in adult bmp7-lacz knock-in  mice at 2 months after birth, a total of  <dig> individual genotypes in adult usag1lacz knock-in  mice at 3 months after birth, and nine usag-1+/+bmp7+/+,  <dig> usag-1+/− bmp7+/+,  <dig> usag-1−/−bmp7+/+,  <dig> usag-1−/−bmp7+/−,  <dig> usag-1+/−bmp7+/−, and  <dig> usag-1+/+bmp7+/− adult f <dig> generation mice at 4 months after birth.
data for volume and cross-sectional area obtained from the right and left incisors were summed.
we measured the cross-sectional area of mandibular supernumerary incisors of three usag-1−/−bmp7+/+ and six usag-1−/−bmp7+/− adult f <dig> generation mice.
we used two usag-1−/−bmp7+/+ and six usag-1−/−bmp7+/− mice at 1 month after birth and one usag-1−/−bmp7+/+ mouse at 4 months after birth.
the total area was measured in cases of multiple supernumerary teeth.
to evaluate the effects of bmp <dig> and usag- <dig> on the total dentition, we also measured mandibular molars.
we obtained photographs of mandibular three molars of the f <dig> generation from directly above the occlusal view under a stereomicroscope and measured the projected area with imagej software using an image of a ruler taken at the same magnification.
the distances between the lens and specimen were fixed.
we examined unilateral mandibular molars from seven usag-1+/+bmp7+/+,  <dig> usag-1+/−bmp7+/+,  <dig> usag-1+/+bmp7+/−, and  <dig> usag-1+/−bmp7+/− adult f <dig> generation mice at 1 month after birth.
because molar form usag-1−/− mice fused  <cit> , we did not measure these molars.
brdu immunostaining
cell proliferation was detected by brdu immunostaining  according to the manufacturer’s specifications.
we made a working solution of brdu in pbs at 1 mg/ml and injected the mice intraperitoneally with 1 ml of the brdu solution.
after 2 h, the mice were sacrificed, and the heads of the embryos were removed and fixed in 4 % paraformaldehyde overnight.
paraffin sections  were then created.
background tissue was stained with hematoxylin for 5 s. we counted approximately 500– <dig> nuclei under a light microscope, and the labeling index was determined as / <dig>
detection of apoptosis
apoptotic cells in situ were detected by the tunel method using an apoptag plus in situ apoptosis detection kit-fluorescein  according to the specifications of the manufacturer.
for paraffin-embedded sections, embryos were fixed in 4 % paraformaldehyde in pbs overnight.
after fluorescent staining, the sections were counterstained with 4′,6-diamidino-2-phenylindole nuclear staining .
statistical analysis
data are presented as the means ± standard deviations.
statistical significance was assessed by analysis of variance  with the statistical program r. gaussian distributions were determined by the anderson darling normality test.
in adult bmp7-lacz knock-in mice,  <dig> wt samples and  <dig> bmp7+/− samples collected from mice at 2 months after birth were analyzed.
we calculated the mean volume and area of the right and left incisors.
statistical significance was determined by unpaired one-tailed t-tests for volume and by mann–whitney u tests for cross-sectional area.
in adult usag1-lacz knock-in mice,  <dig> samples of individual genotypes at 3 months after birth were analyzed as stated above.
statistical significance was determined using kruskal-wallis and steel-dwass tests for multiple comparisons in volume and by one-way anova using a games howell test for multiple comparisons in cross-sectional area.
in the adult f <dig> generation, we examined nine usag-1+/+bmp7+/+,  <dig> usag-1+/−bmp7+/+,  <dig> usag-1−/−bmp7+/+,  <dig> usag-1−/−bmp7+/−,  <dig> usag-1+/−bmp7+/−, and  <dig> usag-1+/+bmp7+/− mice, as mentioned above.
statistical significance was determined by one-way anova using a games howell test for multiple comparisons.
in the analysis of mandibular molars in the adult f <dig> generation, statistical significance was determined by unpaired two-tailed t-tests .
finally, for analysis of cell proliferation using brdu staining in the mandibular incisors in bmp7-lacz knock-in mice at e <dig> and analysis of mandibular supernumerary incisors in the adult f <dig> generation, statistical significance was determined using mann–whitney u tests.
for analysis of cell proliferation using brdu staining in the mandibular incisors in usag1-lacz knock-in mice at e <dig>  statistical significance was determined using kruskal-wallis and steel-dwass tests for multiple comparisons.
RESULTS
expression of bmp <dig> and usag- <dig> at e <dig> and e15
sections from bmp7+/− mice indicated that bmp <dig> was expressed  in the mesenchyme around the tooth germ , rudimentary incisor , and enamel knot .
sections from usag-1+/− mice indicated that usag- <dig> was expressed  in a small portion of the epithelia, excluding the enamel knot and mesenchyme around the tooth germ at e <dig>  during the early bell stage  .fig.
<dig> x-gal staining in mandibular incisors of bmp7+/−  and usag-1+/−  mice at e14/e <dig>  tissue sections from mandibular incisors of bmp7+/−  and usag-1+/−  mice at e <dig> and e <dig> were stained with x-gal .
scale bars indicate 100 μm.
a–f sagittal sections.
g, h coronal sections.
a, b, d, e, g embryos of bmp7+/−  mice.
bmp <dig> was expressed  in the mesenchyme near the tooth germ.
c, f, h embryos of usag-1+/−  mice.
usag- <dig> was expressed  in a small portion of epithelia, except for the enamel knot and the mesenchyme near the tooth germ.
a–c embryonic day  <dig>  d–h embryonic day  <dig>  a, d sections near a rudimentary incisor  with strong expression are shown.
b, e sections near an enamel knot  with strong expression are shown
micro-ct analysis of the mandibular incisors in adult bmp7-lacz knock-in mice
the volume and cross-sectional area of the mandibular incisors were larger in bmp7+/−  mice than in wt mice .
volumes of bmp7+/− mice were significantly larger than those of wt mice .
the cross-sectional areas of bmp7+/− mice were significantly larger than those of wt mice .fig.
<dig> analysis of the adult incisor phenotype.
a micro-ct analysis of mandibular incisors at 2 months after birth.
left to right: image from a stereomicroscope, three-dimensional image, and cross-section.
b micro-ct analysis of mandibular incisors at 3 months after birth.
left to right: image from a stereomicroscope, three-dimensional image, and cross-section.
c left to right: image from a stereomicroscope, three-dimensional image, and cross-section.
d upper images are from a stereomicroscope.
left image is the lingual side.
right image is the buccal side.
lower images show the cross-sectional area of mandibular supernumerary teeth  and mandibular incisors.
a–d scale bars: 1 mm.
e, f volume and cross-sectional area of adult mandibular incisors.
numbers indicate respective incisors.
data in  are the mean volume ± standard deviation  of mandibular incisors at 2 months  or 3 months  after birth.
data in  are the mean cross-sectional area ± sd of mandibular incisors at 2 months  or 3 months  after birth.
g, h volume and cross-sectional area of mandibular incisors in the adult f <dig> generation at 4 months after birth.
data in  are the mean volume ± sd of mandibular incisors.
data in  are the mean cross section ± sd of mandibular incisors.
i statistical analysis of cross-sectional area of mandibular supernumerary incisors in the adult f <dig> generation at 1 month.
error bars: sds.
j images of trimmed and extracted mandibular incisors in a, b, and c. whole computed tomography images of a mouse skull bone and an incisor with soft tissue removed using intage realia are shown .
we opened the image in volume player software  , created .bmp files, and opened the files in image j software .
*p <  <dig> , **p <  <dig> , ***p <  <dig>
micro-ct analysis of the mandibular incisors in adult usag1-lacz knock-in mice
the volume and cross-sectional area of the mandibular incisors in usag-1lacz knock-in  mice were larger than those in any other diplotype .
the volume and cross-sectional area in usag-1−/− mice were significantly larger than those in all other diplotype .
the volume and cross-sectional area in usag-1+/− mice were significantly smaller than those in wt mice .
few mice exhibited malocclusion among usag-1−/− mice.
mice that had malocclusion, taking into account feeding disorders and changes in tooth size, were excluded from the statistical analyses.
two of  <dig> usag-1−/− mice were excluded ,
analysis of the projected areas of the occlusal surface of mandibular molars in adult f <dig> mice
the areas of the occlusal surface of the mandibular molars in bmp7+/− mice were significantly larger than those in wt mice for both the three individual molars and the total .
the areas of the occlusal surface of the mandibular molars in usag-1+/− mice were significantly smaller than those in wt mice, except for m <dig> .fig.
<dig> analysis of the adult molar phenotype.
a occlusal surface of mandibular molars in the f <dig> generation at 1 month after birth.
scale bar indicates 1 mm.
b, c statistical analysis of projected areas of the occlusal surface of three molars and all mandibular molars of the f <dig> generation at 1 month after birth.
ns: not significant, *p <  <dig> , **p <  <dig> , ***p <  <dig> .
b mean area ± standard deviation  of mandibular molars in wt  and bmp7+/−  mice.
c mean area ± standard deviation  of mandibular molars in wt  and usag-1+/−  mice
we used a generalized linear model  to examine the effects of respective genes and genotypes on tooth size.
the model formula of the glm was “tooth size = usag-1 + bmp7”.
we did not consider interactions.
from this analysis, we found that bmp7+/− was associated with an increase in tooth size, whereas usag-1+/− was associated with a decrease in area .
the error structure of the response variable followed a gaussian distribution.
the link function was a linear model.
the explanatory variable was categorical data.
we used genotypes as the categorical data.table  <dig> a generalized linear model to analyze adult tooth size.
the response variable is the total area of the occlusal surface of the molars of the f <dig> generation at 1 month after birth
micro-ct analysis of the mandibular incisors in the adult f <dig> generation
the volume and cross-sectional area of the mandibular incisors of usag-1−/−bmp7+/− mice were larger than those of all other genotypes .
this analysis clarified that the effects of each diplotype of bmp <dig> and usag- <dig> were additive for volume and cross-sectional area .
next, a glm was used, as described in the methods.
usag-1−/− and bmp-7+/− were associated with increased tooth size, whereas usag-1+/− was associated with decreased tooth size .
this analysis confirmed that bmp7+/− was most strongly associated with an increase in tooth size.table  <dig> a generalized linear model to analyze adult tooth size.
the response variables are volume and cross-section of the mandibular incisors of the f <dig> generation at 4 months after birth
two of  <dig> usag-1−/−bmp7+/− mice exhibited malocclusion was excluded ,
analysis of cell proliferation using brdu staining of the mandibular incisors at e15
in murine incisors and molars, tooth eruption and tooth root formation are completed by about p <dig> or p <dig>  the cap stage and early bell stage are considered equal to the morphogenetic phase.
during the early bell stage, only the labial epithelium gives rise to the enamel-forming ameloblasts in mandibular incisors  <cit> .
murine incisors are continuously growing; however, bmp <dig> and usag- <dig> are not expressed in enamel epithelial stem cells in adult mice  <cit> .
therefore, we investigated cell proliferation by brdu staining in the mandibular incisors at e <dig>  during the early bell stage.
the tissues of the mandibular incisors at the early bell stage were categorized as vestibular lamina, stellate reticulum, dental papilla, labial mesenchyme, lingual mesenchyme, labial cervical loop, and lingual cervical loop .
the labeling index of bmp7+/− embryos was significantly higher than that of wt embryos in the cervical loop .
additionally, the labeling index of usag-1−/− embryos was significantly higher than that of wt and usag-1+/− embryos in dental papilla .
the labeling index of wt embryos was significantly higher than that of usag-1+/− embryos in stellate reticulum .fig.
<dig> brdu immunostaining and detection of apoptosis in mandibular incisors at e <dig>  a–j analysis of proliferation using brdu staining in mandibular incisors at e <dig>  a sagittal section of mandibular incisors  of wt mice.
b schematic diagram.
vl: vestibular lamina, sr: stellate reticulum, dp: dental papilla, lam: labial mesenchyme, lim: lingual mesenchyme, lac: labial cervical loop, lic: lingual cervical loop.
c–f sagittal section of cervical loop .
sagittal section of dental papilla .
j analysis of cell proliferation using brdu staining in mandibular incisors of wt and bmp7+/−  mice at e <dig>  percent labeling index in respective tissues of wt and bmp7+/− e <dig> embryos are shown.
labeled cells were counted under 1000× magnification.
we counted approximately  <dig> nuclei under a light microscope and determined the labeling index as:  /  <dig>  data are the mean + standard deviation .
ns: not significant, *p <  <dig>  by mann–whitney u test.
k analysis of cell proliferation using brdu staining in mandibular incisors of usag-1+/+, usag-1+/− and usag- <dig> −/−  mice at e <dig>  percent labeling index of mandibular incisors of usag-1+/+, usag-1+/− and usag-1−/− embryos at e <dig>  data are the mean ± sd.
ns: not significant, *p <  <dig>  by steel dwass test.
l tunel staining in mandibular incisors of bmp7+/− , usag-1−/− , usag-1−/−bmp7+/− , and usag-1+/−  mice at e <dig>  under 100× magnification, the developing bone and cartilage  are shown.
under 200× magnification, meckel’s cartilage , epithelium near the oral cavity , and the mesenchyme near the tooth germ  are shown.
all bars: 100 μm
tunel staining in the mandibular incisors at e15
next, we investigated the occurrence of apoptosis in cells in the mandibular incisors of bmp7+/− , usag-1−/− , usag-1−/−bmp7+/− , and usag-1+/−  embryos at e <dig> .
apoptotic cells were detected near meckel’s cartilage, the developing bone and cartilage, the epithelium near the oral cavity, and the mesenchyme near the tooth germ .
few apoptotic cells were observed in the tooth germ for all genotypes.
in the early bell stage, apoptosis was not associated with the determination of tooth size.
analyzing mandibular supernumerary incisors in the adult f <dig> generation
in the f <dig> generation, the size of supernumerary incisors of usag-1−/−bmp7+/− mice was larger than that of usag-1−/−bmp7+/+ mice .
moreover, the total area of mandibular supernumerary incisors of usag-1−/−bmp7+/− mice was significantly larger than that of usag-1−/−bmp7+/+ mice .
discussion
the expression levels of usag- <dig> and bmp <dig> are opposing in the region near the rudimentary incisor tooth primordia between the maxilla and mandible  <cit> .
moreover, in several types of genetically modified mice, the dental phenotype differs between the maxilla and mandible  <cit> .
for example, in usag-1-deficient mice, supernumerary teeth are observed in 100 % of the maxillary incisor regions, whereas partial penetrance is observed in the mandible  <cit> .
thus, the genetic controls for tooth organ size and shape play critical roles in tooth regeneration.
in this report, we demonstrated that the sizes of mandibular incisor and molar tooth organs in bmp7+/− mice were significantly larger than those of wt mice.
in contrast, the sizes of incisors and molars were significantly smaller in usag-1+/− mice than in wt mice, and the volume and cross-sectional areas in usag-1−/−, mice were significantly larger than those of all other genotypes.
thus, our results demonstrated, for the first time, that bmp <dig> and usag- <dig> heterozygous mice exhibited changes in tooth size.
in conditional bmp7-deficient embryos, the maxillary incisors have been shown to be missing or hypoplastic  <cit> .
thus, the phenotypes of usag- <dig> heterozygous mice and bmp <dig> heterozygous mice were opposite those of usag-1-deficient mice and bmp7-deficient mice, respectively.
our results suggested that the levels of bmp <dig> and usag- <dig> expression in heterozygotes did not reach the threshold level necessary for normal tooth morphogenesis, that is, the embryos exhibited haploinsufficiency.
furthermore, heterozygotes may exhibit changes in downstream gene expression, resulting in changes in tooth size.
global analysis of gene expression in heterozygotes, such as microarray analysis and next-generation sequencing, is needed in order to elucidate the molecular mechanism involved in these processes.
the expression level of bmp <dig> was similar to those of wnt5a and mrfzb <dig> and was localized adjacent to usag- <dig> expression in the dental mesenchyme.
usag- <dig> is a bmp <dig> antagonist; however, the effects of bmp <dig> and usag- <dig> were additive within the mandibular incisors of the f <dig> generation.
brdu assays confirmed that cell proliferation was increased within the cervical loop in association with larger sized mandibular incisors in bmp7+/− embryos.
furthermore, we confirmed that the increased cell proliferation of dental papilla mesenchymal cells was associated with larger mandibular incisors in usag-1−/− embryos.
importantly, apoptosis was not associated with tooth size at e <dig>  these data indicated that there was a difference in the mechanisms of action of bmp <dig> and usag- <dig> during the cap or bell stages of tooth morphogenesis.
continuous growth and enamel deposition in incisors can be modulated by the levels of fgf3/ <dig>  activin, and bmp2/4/ <dig> mesenchymally expressed in the epithelial stem cell niche  <cit> .
thus, we concluded that elongation of the cervical loop in bmp7+/− embryos at e <dig> enlarged the incisors and that bmp <dig> expressed in the mesenchyme around the cervical loop had distinct local effects on the loop.
the volume and cross-sectional area were largest in usag-1−/− mice, and the labeling index of the dental papilla in usag-1−/− mice was highest of all genotypes examined.
we have also reported that phosphorylated smad1/5/ <dig> levels are increased and that β-catenin is localized in the nucleus in odontogenic mesenchymal cells within the maxillary rudimentary incisor tooth organ in usag-1-deficient embryos  <cit> .
using organ culture of wt and usag-1-deficient mandibular incisors, munne et al.
demonstrated that usag- <dig> expression is limited to the mesenchyme and that the dental mesenchyme may limit supernumerary tooth induction resulting from activated wnt signaling, thereby minimizing the amount of mesenchymal tissue surrounding the incisor tooth germs prior to culture  <cit> .
usag- <dig> is downstream of sonic hedgehog  signaling; therefore, a wnt-shh-sostdc <dig> negative feedback loop may control the spatial patterning of teeth, and wnt, shh, and sostdc <dig> may act as the activator, mediator, and inhibitor, respectively, in reaction–diffusion models  <cit> .
consistent with this, patients with supernumerary teeth have larger teeth than controls in humans  <cit> .
thus, these findings, combined with our new results, suggest that enhanced bmp- and wnt-mediated signal transduction in the dental mesenchyme of usag-1−/− mice may increase the proliferation of cells in the dental mesenchyme at the cap or early bell stages, resulting in increased tooth size and formation of supernumerary teeth.
the size of a tooth is determined not only by genes related to tooth development but also by the development of the mandible.
the size of mandibular incisors is altered by craniosynostoss in the apert fgfr2s252w mouse model  <cit> .
importantly, in our study, we found no significant difference in the linear distance of the anteroposterior diameter and altitude between respective genotypes in bmp7-lacz knock-in mice at 2 months of age .
however, at 3 months of age, usag-1−/− mice exhibited a significantly longer linear distance of the anteroposterior diameter and altitude compared with that in wt and usag-1+/− mice .
the development of the mandible in the context of usag- <dig> deficiency may be associated with changes in the size of the mandibular incisor and molar in usag-1−/− mice.
these results suggested that we may be able to control the size of teeth by regulating the gene expression level locally in humans when dental epithelial stem cells, such as third dentitions and outer enamel epithelium, are used for tooth regeneration in vivo.
CONCLUSIONS
our findings showed that bmp <dig> heterozygous mice exhibited dramatic increases in tooth size and that tooth size was controlled by the expression levels of specific genes.
ethics approval and consent to participate
the study protocol and procedures were approved by the animal research committee of kyoto university  and the recombinant dna experiment safety committee of kyoto university.
all experiments were carried out in accordance with the approved guidelines.
consent for publication
not applicable.
additional files
additional file 1: figure s <dig>  x-gal staining in mandibular molars of usag-1+/−  mice at e <dig> and e <dig>  tissue sections from mandibular molars of usag-1+/−  mice at e <dig> and e <dig> were stained with x-gal.
scale bar: 100 μm.
sagittal sections.
coronal sections.
usag- <dig> was expressed  in a small portion of epithelia, except for the enamel knot and the mesenchyme near the tooth germ.
additional file 2: figure s <dig>  malocclusion observed in usag-1−/−  mice and usag-1−/− bmp7+/−  mice.
normal occlusion.
a usag-1−/− female mouse in the c57bl/ <dig> background at 3 months after birth.
these data were added in the analysis of the lower incisors.
scale bar: 1 mm.
normal occlusion.
a usag-1−/−bmp7+/− male mouse in the f <dig> generation at 4 months after birth.
these data were added in the analysis of the lower incisors.
malocclusion.
a usag-1−/− female mouse in c57bl/ <dig> background at 3 months after birth.
the mouse was excluded from the analysis of the lower incisors.
malocclusion.
a usag-1−/−bmp7+/− female mouse in the f <dig> generation at 4 months after birth.
the mouse was excluded from the analysis of the lower incisors.
additional file 3: figure s <dig>  difference in mandibular morphology between respective genotypes in bmp <dig> or usag-1-lacz knock-in mice.
wild-type mouse  mandible at 2 months after birth with locations of landmarks used to analyze the morphological differences between respective genotypes.
linear distances between the identifiable landmarks were measured.
a: inferior-most point on the incisor alveolar rim, b: anterior point on the molar alveolar rim, c: inferior-most point on border of the ramus inferior to incisor alveolar, d: mandibular angle.
difference in mandibular morphology between wt and bmp7+/− mice.
we analyzed a total of  <dig> wt and  <dig> bmp7+/− samples in adult bmp7-lacz knock-in  mice at 2 months after birth.
statistical significance was determined by the mann–whitney u test.
difference in the mandibular morphology among wt, usag-1+/−, and usag-1−/− mice.
we analyzed a total of  <dig> individual genotypes in adult usag1-lacz knock-in  mice at 3 months after birth.
statistical significance was determined using a kruskal-wallis test and a steel-dwass test for multiple comparisons.
*p <  <dig> .
abbreviations
−/−knockout genotype
+/−heterozygous genotype
+/+wild-type
lacz/laczknockout genotype in lacz knock-in mice
wtwild-type
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ks, kt, kb, ma, and mm contributed to the conception and design of the experiment.
ks, hk, yt, and ht contributed to acquisition of the data.
ma carried out measurement and analysis of the molars.
ks drafted the manuscript.
kt, ma, bh, hcs, ms, and as revised the manuscript.
all authors reviewed the manuscript.
all authors read and approved the final manuscript.
bmp7-lacz knock-in mice were a kind gift from dr. elizabeth robertson of oxford university.
the authors are indebted to dr. motoko yanagita of kyoto university for helpful discussions.
usag-1-lacz knock-in mice were a kind gift from aris n. economides of regeneron pharmaceuticals inc. in tarrytown, new york, usa.
funding
this study was supported by a grant-in-aid for scientific research  from the japanese society for the promotion of science.

 development of the dentition is regulated by time- and position-specific reciprocal epithelial-mesenchymal interactions. these odontogenic interactions are directed and coordinated by transcription factors, growth and signaling factors, their cognate receptors, and extracellular matrix constituents. collectively, various combinations of molecules within these interactions determine when or where teeth develop and modulate the specifications for tooth size and shape. noggin, wnt5a, and mfrzb genes have been shown to regulate tooth size. cai et al., using hetero-specific tissue recombination from rat molar tooth organs, demonstrated that tooth size is determined not by dental papilla mesenchymal cell number, but by intrinsic tissue-specific dental papilla mesenchymal factors. in addition, exogenous treatment with bone morphogenic protein noggin, fibroblast growth factor and fgf does not affect tooth size, despite the observation that bmp2/ fgf3/ lef and wnt5a/5b are expressed in the dental mesenchyme during the cap and early bell stages of tooth morphogenesis. the crown width of a bioengineered molar reconstructed with dissociated epithelial and mesenchymal cells through the organ germ method is correlated with the length of the contact area between the epithelial and mesenchymal cell layers. in conditional bmp7-deficient embryos, the maxillary incisor tooth organs are either missing or hypoplastic, and the development of the first molar tooth organs is delayed, malformed, or missing. therefore, bmp is assumed to be an essential growth factor for tooth morphogenesis. bmp is a 35-kda homodimeric protein that is associated with a variety of signaling pathways, including the canonical smad pathway, mitogen-activated protein kinase -related pathways, and the phosphoinositol 3-kinase /akt pathway. uterine sensitization-associated gene- is a heavily glycosylated 28–30 kda secretory protein that functions as a monomer to induce signal transduction. usag- binds to the wnt coreceptors low-density lipoprotein receptor-related protein and lrp and inhibits wnt signaling. lrp modulates and integrates bmp and canonical wnt signaling during tooth morphogenesis by binding to secreted usag-. usag- expression is further restricted to the distal renal tubules, in a pattern similar to the localization of bmp usag- is a bmp antagonist; it interacts with bmp in the developing and adult kidney and directly binds to bmp2/4/ as assayed using co-immunoprecipitation studies. moreover, we previously reported that usag-1-deficient mice exhibit supernumerary maxillary incisors in response to enhanced bmp signaling and that bmp signaling is modulated by wnt signaling in usag-1-deficient mice. based on these observations and findings, we hypothesized that bmp and usag- play important roles in tooth morphogenesis. therefore, we previously established double genetically modified mice to analyze the in vivo inter-relationships between bmp and usag- using this model, we demonstrated that usag- inhibits bmp signaling, leading to apoptosis and degeneration of rudimentary tooth germs in maxillary supernumerary incisor formation. moreover, we found that the tooth size in mandibular incisors of bmp heterozygous mice was altered. accordingly, in this study, we examined the effects of bmp and usag- signaling on tooth size in mandibular incisors using the f generation of mice. methods production and analysis of usag-1- and bmp7-lacz knock-in mice bmp7-lacz knock-in mice and usag1-lacz knock-in mice were produced as previously described. bmp7-deficient mice were embryonic lethal. day e was established as midnight prior to finding a vaginal plug. polymerase chain reaction amplification was performed using kod fx neo polymerase and specific primers for genotyping. x-gal staining the bacterial lacz gene in bmp7lacz/lacz and usag1lacz/lacz mice was knocked into the gene of interest. frozen sections of embryos were fixed in 4 % paraformaldehyde for 2 min and stained with x-gal twice as whole mounts and frozen sections, followed by counterstaining with nuclear fast red. for x-gal staining, embryos were incubated at 37 °c in buffer 35 mm k4fe 2 mm mgcl  % np-  % sodium deoxycholate, 1× phosphate-buffered saline ). we observed mandibular incisors in bmp7+/− and usag-1+/− mice and mandibular molars in usag-1+/− mice at e and e analysis of the adult tooth phenotype three-dimensional computed tomography scans were performed using the heads of adult mice. we converted cb files to tiff files. cb files had 512 × 512 pixels, bits, and a voxel size of x:y:z = 1:1:. next, 3d images were reconstructed and analyzed with computer imaging software. the mandibular incisor volume was measured using the imaging software; then, to measure the cross-sectional area, mandibular incisors were cut vertically to the tooth axes at the uppermost point on the incisor alveolar rim, so as to avoid the influence of environmental factors, such as dental occlusion and tooth attrition. the cross-sectional area of the incisors was measured using imagej software. we analyzed a total of wild-type and bmp7+/− samples in adult bmp7-lacz knock-in mice at 2 months after birth, a total of individual genotypes in adult usag1lacz knock-in mice at 3 months after birth, and nine usag-1+/+bmp7+/+, usag-1+/− bmp7+/+, usag-1−/−bmp7+/+, usag-1−/−bmp7+/−, usag-1+/−bmp7+/−, and usag-1+/+bmp7+/− adult f generation mice at 4 months after birth. data for volume and cross-sectional area obtained from the right and left incisors were summed. we measured the cross-sectional area of mandibular supernumerary incisors of three usag-1−/−bmp7+/+ and six usag-1−/−bmp7+/− adult f generation mice. we used two usag-1−/−bmp7+/+ and six usag-1−/−bmp7+/− mice at 1 month after birth and one usag-1−/−bmp7+/+ mouse at 4 months after birth. the total area was measured in cases of multiple supernumerary teeth. to evaluate the effects of bmp and usag- on the total dentition, we also measured mandibular molars. we obtained photographs of mandibular three molars of the f generation from directly above the occlusal view under a stereomicroscope and measured the projected area with imagej software using an image of a ruler taken at the same magnification. the distances between the lens and specimen were fixed. we examined unilateral mandibular molars from seven usag-1+/+bmp7+/+, usag-1+/−bmp7+/+, usag-1+/+bmp7+/−, and usag-1+/−bmp7+/− adult f generation mice at 1 month after birth. because molar form usag-1−/− mice fused, we did not measure these molars. brdu immunostaining cell proliferation was detected by brdu immunostaining according to the manufacturer’s specifications. we made a working solution of brdu in pbs at 1 mg/ml and injected the mice intraperitoneally with 1 ml of the brdu solution. after 2 h, the mice were sacrificed, and the heads of the embryos were removed and fixed in 4 % paraformaldehyde overnight. paraffin sections were then created. background tissue was stained with hematoxylin for 5 s. we counted approximately 500– nuclei under a light microscope, and the labeling index was determined as / detection of apoptosis apoptotic cells in situ were detected by the tunel method using an apoptag plus in situ apoptosis detection kit-fluorescein according to the specifications of the manufacturer. for paraffin-embedded sections, embryos were fixed in 4 % paraformaldehyde in pbs overnight. after fluorescent staining, the sections were counterstained with 4′,6-diamidino-2-phenylindole nuclear staining. statistical analysis data are presented as the means ± standard deviations. statistical significance was assessed by analysis of variance with the statistical program r. gaussian distributions were determined by the anderson darling normality test. in adult bmp7-lacz knock-in mice, wt samples and bmp7+/− samples collected from mice at 2 months after birth were analyzed. we calculated the mean volume and area of the right and left incisors. statistical significance was determined by unpaired one-tailed t-tests for volume and by mann–whitney u tests for cross-sectional area. in adult usag1-lacz knock-in mice, samples of individual genotypes at 3 months after birth were analyzed as stated above. statistical significance was determined using kruskal-wallis and steel-dwass tests for multiple comparisons in volume and by one-way anova using a games howell test for multiple comparisons in cross-sectional area. in the adult f generation, we examined nine usag-1+/+bmp7+/+, usag-1+/−bmp7+/+, usag-1−/−bmp7+/+, usag-1−/−bmp7+/−, usag-1+/−bmp7+/−, and usag-1+/+bmp7+/− mice, as mentioned above. statistical significance was determined by one-way anova using a games howell test for multiple comparisons. in the analysis of mandibular molars in the adult f generation, statistical significance was determined by unpaired two-tailed t-tests. finally, for analysis of cell proliferation using brdu staining in the mandibular incisors in bmp7-lacz knock-in mice at e and analysis of mandibular supernumerary incisors in the adult f generation, statistical significance was determined using mann–whitney u tests. for analysis of cell proliferation using brdu staining in the mandibular incisors in usag1-lacz knock-in mice at e statistical significance was determined using kruskal-wallis and steel-dwass tests for multiple comparisons.  expression of bmp and usag- at e and e15 sections from bmp7+/− mice indicated that bmp was expressed in the mesenchyme around the tooth germ, rudimentary incisor, and enamel knot. sections from usag-1+/− mice indicated that usag- was expressed in a small portion of the epithelia, excluding the enamel knot and mesenchyme around the tooth germ at e during the early bell stage.fig. x-gal staining in mandibular incisors of bmp7+/− and usag-1+/− mice at e14/e tissue sections from mandibular incisors of bmp7+/− and usag-1+/− mice at e and e were stained with x-gal. scale bars indicate 100 μm. a–f sagittal sections. g, h coronal sections. a, b, d, e, g embryos of bmp7+/− mice. bmp was expressed in the mesenchyme near the tooth germ. c, f, h embryos of usag-1+/− mice. usag- was expressed in a small portion of epithelia, except for the enamel knot and the mesenchyme near the tooth germ. a–c embryonic day d–h embryonic day a, d sections near a rudimentary incisor with strong expression are shown. b, e sections near an enamel knot with strong expression are shown micro-ct analysis of the mandibular incisors in adult bmp7-lacz knock-in mice the volume and cross-sectional area of the mandibular incisors were larger in bmp7+/− mice than in wt mice. volumes of bmp7+/− mice were significantly larger than those of wt mice. the cross-sectional areas of bmp7+/− mice were significantly larger than those of wt mice.fig. analysis of the adult incisor phenotype. a micro-ct analysis of mandibular incisors at 2 months after birth. left to right: image from a stereomicroscope, three-dimensional image, and cross-section. b micro-ct analysis of mandibular incisors at 3 months after birth. left to right: image from a stereomicroscope, three-dimensional image, and cross-section. c left to right: image from a stereomicroscope, three-dimensional image, and cross-section. d upper images are from a stereomicroscope. left image is the lingual side. right image is the buccal side. lower images show the cross-sectional area of mandibular supernumerary teeth and mandibular incisors. a–d scale bars: 1 mm. e, f volume and cross-sectional area of adult mandibular incisors. numbers indicate respective incisors. data in are the mean volume ± standard deviation of mandibular incisors at 2 months or 3 months after birth. data in are the mean cross-sectional area ± sd of mandibular incisors at 2 months or 3 months after birth. g, h volume and cross-sectional area of mandibular incisors in the adult f generation at 4 months after birth. data in are the mean volume ± sd of mandibular incisors. data in are the mean cross section ± sd of mandibular incisors. i statistical analysis of cross-sectional area of mandibular supernumerary incisors in the adult f generation at 1 month. error bars: sds. j images of trimmed and extracted mandibular incisors in a, b, and c. whole computed tomography images of a mouse skull bone and an incisor with soft tissue removed using intage realia are shown. we opened the image in volume player software, created.bmp files, and opened the files in image j software. *p < , **p < , ***p <  micro-ct analysis of the mandibular incisors in adult usag1-lacz knock-in mice the volume and cross-sectional area of the mandibular incisors in usag-1lacz knock-in mice were larger than those in any other diplotype. the volume and cross-sectional area in usag-1−/− mice were significantly larger than those in all other diplotype. the volume and cross-sectional area in usag-1+/− mice were significantly smaller than those in wt mice. few mice exhibited malocclusion among usag-1−/− mice. mice that had malocclusion, taking into account feeding disorders and changes in tooth size, were excluded from the statistical analyses. two of usag-1−/− mice were excluded, analysis of the projected areas of the occlusal surface of mandibular molars in adult f mice the areas of the occlusal surface of the mandibular molars in bmp7+/− mice were significantly larger than those in wt mice for both the three individual molars and the total. the areas of the occlusal surface of the mandibular molars in usag-1+/− mice were significantly smaller than those in wt mice, except for m.fig. analysis of the adult molar phenotype. a occlusal surface of mandibular molars in the f generation at 1 month after birth. scale bar indicates 1 mm. b, c statistical analysis of projected areas of the occlusal surface of three molars and all mandibular molars of the f generation at 1 month after birth. ns: not significant, *p < , **p < , ***p < . b mean area ± standard deviation of mandibular molars in wt and bmp7+/− mice. c mean area ± standard deviation of mandibular molars in wt and usag-1+/− mice we used a generalized linear model to examine the effects of respective genes and genotypes on tooth size. the model formula of the glm was “tooth size = usag-1 + bmp7”. we did not consider interactions. from this analysis, we found that bmp7+/− was associated with an increase in tooth size, whereas usag-1+/− was associated with a decrease in area. the error structure of the response variable followed a gaussian distribution. the link function was a linear model. the explanatory variable was categorical data. we used genotypes as the categorical data.table a generalized linear model to analyze adult tooth size. the response variable is the total area of the occlusal surface of the molars of the f generation at 1 month after birth micro-ct analysis of the mandibular incisors in the adult f generation the volume and cross-sectional area of the mandibular incisors of usag-1−/−bmp7+/− mice were larger than those of all other genotypes. this analysis clarified that the effects of each diplotype of bmp and usag- were additive for volume and cross-sectional area. next, a glm was used, as described in the methods. usag-1−/− and bmp-7+/− were associated with increased tooth size, whereas usag-1+/− was associated with decreased tooth size. this analysis confirmed that bmp7+/− was most strongly associated with an increase in tooth size.table a generalized linear model to analyze adult tooth size. the response variables are volume and cross-section of the mandibular incisors of the f generation at 4 months after birth two of usag-1−/−bmp7+/− mice exhibited malocclusion was excluded, analysis of cell proliferation using brdu staining of the mandibular incisors at e15 in murine incisors and molars, tooth eruption and tooth root formation are completed by about p or p the cap stage and early bell stage are considered equal to the morphogenetic phase. during the early bell stage, only the labial epithelium gives rise to the enamel-forming ameloblasts in mandibular incisors. murine incisors are continuously growing; however, bmp and usag- are not expressed in enamel epithelial stem cells in adult mice. therefore, we investigated cell proliferation by brdu staining in the mandibular incisors at e during the early bell stage. the tissues of the mandibular incisors at the early bell stage were categorized as vestibular lamina, stellate reticulum, dental papilla, labial mesenchyme, lingual mesenchyme, labial cervical loop, and lingual cervical loop. the labeling index of bmp7+/− embryos was significantly higher than that of wt embryos in the cervical loop. additionally, the labeling index of usag-1−/− embryos was significantly higher than that of wt and usag-1+/− embryos in dental papilla. the labeling index of wt embryos was significantly higher than that of usag-1+/− embryos in stellate reticulum.fig. brdu immunostaining and detection of apoptosis in mandibular incisors at e a–j analysis of proliferation using brdu staining in mandibular incisors at e a sagittal section of mandibular incisors of wt mice. b schematic diagram. vl: vestibular lamina, sr: stellate reticulum, dp: dental papilla, lam: labial mesenchyme, lim: lingual mesenchyme, lac: labial cervical loop, lic: lingual cervical loop. c–f sagittal section of cervical loop. sagittal section of dental papilla. j analysis of cell proliferation using brdu staining in mandibular incisors of wt and bmp7+/− mice at e percent labeling index in respective tissues of wt and bmp7+/− e embryos are shown. labeled cells were counted under 1000× magnification. we counted approximately nuclei under a light microscope and determined the labeling index as: / data are the mean + standard deviation. ns: not significant, *p <  by mann–whitney u test. k analysis of cell proliferation using brdu staining in mandibular incisors of usag-1+/+, usag-1+/− and usag- −/− mice at e percent labeling index of mandibular incisors of usag-1+/+, usag-1+/− and usag-1−/− embryos at e data are the mean ± sd. ns: not significant, *p <  by steel dwass test. l tunel staining in mandibular incisors of bmp7+/−, usag-1−/−, usag-1−/−bmp7+/−, and usag-1+/− mice at e under 100× magnification, the developing bone and cartilage are shown. under 200× magnification, meckel’s cartilage, epithelium near the oral cavity, and the mesenchyme near the tooth germ are shown. all bars: 100 μm tunel staining in the mandibular incisors at e15 next, we investigated the occurrence of apoptosis in cells in the mandibular incisors of bmp7+/−, usag-1−/−, usag-1−/−bmp7+/−, and usag-1+/− embryos at e. apoptotic cells were detected near meckel’s cartilage, the developing bone and cartilage, the epithelium near the oral cavity, and the mesenchyme near the tooth germ. few apoptotic cells were observed in the tooth germ for all genotypes. in the early bell stage, apoptosis was not associated with the determination of tooth size. analyzing mandibular supernumerary incisors in the adult f generation in the f generation, the size of supernumerary incisors of usag-1−/−bmp7+/− mice was larger than that of usag-1−/−bmp7+/+ mice. moreover, the total area of mandibular supernumerary incisors of usag-1−/−bmp7+/− mice was significantly larger than that of usag-1−/−bmp7+/+ mice. discussion the expression levels of usag- and bmp are opposing in the region near the rudimentary incisor tooth primordia between the maxilla and mandible. moreover, in several types of genetically modified mice, the dental phenotype differs between the maxilla and mandible. for example, in usag-1-deficient mice, supernumerary teeth are observed in 100 % of the maxillary incisor regions, whereas partial penetrance is observed in the mandible. thus, the genetic controls for tooth organ size and shape play critical roles in tooth regeneration. in this report, we demonstrated that the sizes of mandibular incisor and molar tooth organs in bmp7+/− mice were significantly larger than those of wt mice. in contrast, the sizes of incisors and molars were significantly smaller in usag-1+/− mice than in wt mice, and the volume and cross-sectional areas in usag-1−/−, mice were significantly larger than those of all other genotypes. thus, our results demonstrated, for the first time, that bmp and usag- heterozygous mice exhibited changes in tooth size. in conditional bmp7-deficient embryos, the maxillary incisors have been shown to be missing or hypoplastic. thus, the phenotypes of usag- heterozygous mice and bmp heterozygous mice were opposite those of usag-1-deficient mice and bmp7-deficient mice, respectively. our results suggested that the levels of bmp and usag- expression in heterozygotes did not reach the threshold level necessary for normal tooth morphogenesis, that is, the embryos exhibited haploinsufficiency. furthermore, heterozygotes may exhibit changes in downstream gene expression, resulting in changes in tooth size. global analysis of gene expression in heterozygotes, such as microarray analysis and next-generation sequencing, is needed in order to elucidate the molecular mechanism involved in these processes. the expression level of bmp was similar to those of wnt5a and mrfzb and was localized adjacent to usag- expression in the dental mesenchyme. usag- is a bmp antagonist; however, the effects of bmp and usag- were additive within the mandibular incisors of the f generation. brdu assays confirmed that cell proliferation was increased within the cervical loop in association with larger sized mandibular incisors in bmp7+/− embryos. furthermore, we confirmed that the increased cell proliferation of dental papilla mesenchymal cells was associated with larger mandibular incisors in usag-1−/− embryos. importantly, apoptosis was not associated with tooth size at e these data indicated that there was a difference in the mechanisms of action of bmp and usag- during the cap or bell stages of tooth morphogenesis. continuous growth and enamel deposition in incisors can be modulated by the levels of fgf3/ activin, and bmp2/4/ mesenchymally expressed in the epithelial stem cell niche. thus, we concluded that elongation of the cervical loop in bmp7+/− embryos at e enlarged the incisors and that bmp expressed in the mesenchyme around the cervical loop had distinct local effects on the loop. the volume and cross-sectional area were largest in usag-1−/− mice, and the labeling index of the dental papilla in usag-1−/− mice was highest of all genotypes examined. we have also reported that phosphorylated smad1/5/ levels are increased and that β-catenin is localized in the nucleus in odontogenic mesenchymal cells within the maxillary rudimentary incisor tooth organ in usag-1-deficient embryos. using organ culture of wt and usag-1-deficient mandibular incisors, munne et al. demonstrated that usag- expression is limited to the mesenchyme and that the dental mesenchyme may limit supernumerary tooth induction resulting from activated wnt signaling, thereby minimizing the amount of mesenchymal tissue surrounding the incisor tooth germs prior to culture. usag- is downstream of sonic hedgehog signaling; therefore, a wnt-shh-sostdc negative feedback loop may control the spatial patterning of teeth, and wnt, shh, and sostdc may act as the activator, mediator, and inhibitor, respectively, in reaction–diffusion models. consistent with this, patients with supernumerary teeth have larger teeth than controls in humans. thus, these findings, combined with our new results, suggest that enhanced bmp- and wnt-mediated signal transduction in the dental mesenchyme of usag-1−/− mice may increase the proliferation of cells in the dental mesenchyme at the cap or early bell stages, resulting in increased tooth size and formation of supernumerary teeth. the size of a tooth is determined not only by genes related to tooth development but also by the development of the mandible. the size of mandibular incisors is altered by craniosynostoss in the apert fgfr2s252w mouse model. importantly, in our study, we found no significant difference in the linear distance of the anteroposterior diameter and altitude between respective genotypes in bmp7-lacz knock-in mice at 2 months of age. however, at 3 months of age, usag-1−/− mice exhibited a significantly longer linear distance of the anteroposterior diameter and altitude compared with that in wt and usag-1+/− mice. the development of the mandible in the context of usag- deficiency may be associated with changes in the size of the mandibular incisor and molar in usag-1−/− mice. these results suggested that we may be able to control the size of teeth by regulating the gene expression level locally in humans when dental epithelial stem cells, such as third dentitions and outer enamel epithelium, are used for tooth regeneration in vivo.  our findings showed that bmp heterozygous mice exhibited dramatic increases in tooth size and that tooth size was controlled by the expression levels of specific genes. ethics approval and consent to participate the study protocol and procedures were approved by the animal research committee of kyoto university and the recombinant dna experiment safety committee of kyoto university. all experiments were carried out in accordance with the approved guidelines. consent for publication not applicable. additional files additional file 1: figure s x-gal staining in mandibular molars of usag-1+/− mice at e and e tissue sections from mandibular molars of usag-1+/− mice at e and e were stained with x-gal. scale bar: 100 μm. sagittal sections. coronal sections. usag- was expressed in a small portion of epithelia, except for the enamel knot and the mesenchyme near the tooth germ. additional file 2: figure s malocclusion observed in usag-1−/− mice and usag-1−/− bmp7+/− mice. normal occlusion. a usag-1−/− female mouse in the c57bl/ background at 3 months after birth. these data were added in the analysis of the lower incisors. scale bar: 1 mm. normal occlusion. a usag-1−/−bmp7+/− male mouse in the f generation at 4 months after birth. these data were added in the analysis of the lower incisors. malocclusion. a usag-1−/− female mouse in c57bl/ background at 3 months after birth. the mouse was excluded from the analysis of the lower incisors. malocclusion. a usag-1−/−bmp7+/− female mouse in the f generation at 4 months after birth. the mouse was excluded from the analysis of the lower incisors. additional file 3: figure s difference in mandibular morphology between respective genotypes in bmp or usag-1-lacz knock-in mice. wild-type mouse mandible at 2 months after birth with locations of landmarks used to analyze the morphological differences between respective genotypes. linear distances between the identifiable landmarks were measured. a: inferior-most point on the incisor alveolar rim, b: anterior point on the molar alveolar rim, c: inferior-most point on border of the ramus inferior to incisor alveolar, d: mandibular angle. difference in mandibular morphology between wt and bmp7+/− mice. we analyzed a total of wt and bmp7+/− samples in adult bmp7-lacz knock-in mice at 2 months after birth. statistical significance was determined by the mann–whitney u test. difference in the mandibular morphology among wt, usag-1+/−, and usag-1−/− mice. we analyzed a total of individual genotypes in adult usag1-lacz knock-in mice at 3 months after birth. statistical significance was determined using a kruskal-wallis test and a steel-dwass test for multiple comparisons. *p < . abbreviations −/−knockout genotype +/−heterozygous genotype +/+wild-type lacz/laczknockout genotype in lacz knock-in mice wtwild-type competing interests the authors declare that they have no competing interests. authors’ contributions ks, kt, kb, ma, and mm contributed to the conception and design of the experiment. ks, hk, yt, and ht contributed to acquisition of the data. ma carried out measurement and analysis of the molars. ks drafted the manuscript. kt, ma, bh, hcs, ms, and as revised the manuscript. all authors reviewed the manuscript. all authors read and approved the final manuscript. bmp7-lacz knock-in mice were a kind gift from dr. elizabeth robertson of oxford university. the authors are indebted to dr. motoko yanagita of kyoto university for helpful discussions. usag-1-lacz knock-in mice were a kind gift from aris n. economides of regeneron pharmaceuticals inc. in tarrytown, new york, usa. funding this study was supported by a grant-in-aid for scientific research from the japanese society for the promotion of science.
in the f generation, the effects of these genes were additive; bmp7+/− was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of usag-1−/−bmp7+/− mice was significantly larger than that of usag-1−/−bmp +/+ mice. thus, we hypothesized that bmp and usag- signaling molecules may play important roles in tooth morphogenesis. wnt5a and mrfzb genes are involved in the regulation of tooth size, and their expression levels are similar to that of bmp during morphogenesis, including during the cap and early bell stages of tooth formation. we measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography in adult bmp7- and usag-1-lacz knock-in mice and their f generation upon interbreeding. tooth sizebmp7usag-1mouse modeltooth volumetooth morphogenesisissue-copyright-statement© the author 2016 at embryonic day, brdu assays demonstrated that the labeling index of bmp7+/− embryos was significantly higher than that of wt embryos in the cervical loop. additionally, the labeling index of usag-1−/− embryos was significantly the highest of all genotypes examined in dental papilla. bmp heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. the mandibular incisors of adult bmp7+/− mice were significantly larger than those of wild-type mice. the mandibular incisors of adult usag-1−/− mice were the largest of all genotypes examined. in this study, we established double genetically modified mice to examine the in vivo inter-relationships between bmp and usag- 
 wnt5a and mrfzb genes are involved in the regulation of tooth size, and their expression levels are similar to that of bmp during morphogenesis, including during the cap and early bell stages of tooth formation. we previously reported that usag-1-deficient mice form supernumerary maxillary incisors. thus, we hypothesized that bmp and usag- signaling molecules may play important roles in tooth morphogenesis. in this study, we established double genetically modified mice to examine the in vivo inter-relationships between bmp and usag-  we measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography in adult bmp7- and usag-1-lacz knock-in mice and their f generation upon interbreeding. the mandibular incisors of adult bmp7+/− mice were significantly larger than those of wild-type mice. the mandibular incisors of adult usag-1−/− mice were the largest of all genotypes examined. in the f generation, the effects of these genes were additive; bmp7+/− was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of usag-1−/−bmp7+/− mice was significantly larger than that of usag-1−/−bmp +/+ mice. at embryonic day, brdu assays demonstrated that the labeling index of bmp7+/− embryos was significantly higher than that of wt embryos in the cervical loop. additionally, the labeling index of usag-1−/− embryos was significantly the highest of all genotypes examined in dental papilla.  bmp heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. our findings may be useful in applications of regenerative medicine and dentistry. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tooth sizebmp7usag-1mouse modeltooth volumetooth morphogenesisissue-copyright-statement© the author 2016 