BACKGROUND
odontogenesis is driven by many genes encoding signature and signaling molecules, which are regulated by epithelial-mesenchymal interactions mediated by the fine-tuning of conserved signaling pathways including shh, wnt, fgf, tgf-β, bmp, eda, etc.
<cit> .
the current understanding of the molecular mechanisms controlling tooth development has come mostly from studies in mice, which have only one set of non-replaced dentition with a diastema and are obviously different from humans with respect to tooth anatomy and development; therefore, relatively little is known about the mechanisms of tooth replacement in mammals  <cit> .
a desirable model remains a significant obstacle for understanding the mechanisms of tooth replacement.
pigs resemble humans in anatomy, physiology, pathophysiology, development, and immune responses  <cit> , and have been successfully used as an experimental model for craniofacial research  <cit> .
recently, swine have become more popular as a useful pre-clinical model for jaw osteoradionecrosis, jaw bone defects, salivary gland radiation damage, periodontal diseases, craniofacial disorders, temporal mandibular joint fractures, and autoimmune diseases  <cit> .
swine would serve as excellent pre-clinical experiment alternatives for tooth development and regeneration compared with the rodent models widely used.
the initiation, eruption time, and sequence of tooth development in miniature pigs are quite similar in humans.
in addition, swine have diphyodont dentition, which is an excellent model for studying teeth replacement  <cit> .
the teeth anatomy and deciduous teeth development of miniature pigs have been described previously  <cit> .
to date, there is a lack of gene expression and regulation profiles during odontogenesis in swine and the differences in gene expression profiles between swine, rodents, and human remain largely undefined.
recently, the porcine genome project was completed  <cit> ; thus, further analysis of the mechanisms of morphogenetic patterns and diphyodont replacement should be possible using molecular methods in this large animal model.
the primary teeth and successional counterparts of wuzhishan miniature pigs have many similarities to humans in size, morphology, number, and form of teeth  <cit> .
we previously determined the patterns of early diphyodont morphogenesis in the wuzhishan miniature pig  <cit> .
the results indicated that the last deciduous molar  in the mandible of the miniature pig undergoes cap stage at embryonic day  <dig> , bell stage at e <dig>  secretory stage at e <dig>  and its successional tooth is derived from the lingual successional dental lamina , which is visible at e <dig> when its deciduous counterpart is in bell stage, and changes little in morphology at e <dig> .
the developmental features offer a desirable pattern for understanding diphyodont replacement.
moreover, the analysis of the genome sequence of the wuzhishan miniature pig has been published  <cit> .
based on the above studies, we used the wuzhishan miniature pig as a large animal model to investigate the gene expression profiles and functional regulation networks of developing deciduous molars and their replacement counterparts using microarrays.
we analyzed the differential gene expression profiles and characterized the gene regulatory networks of morphogenesis and cell differentiation during early diphyodont morphogenesis.
we identified the stage-specific genes and pathways with differential regulation.
the results provide a molecular base for further research on tooth development, replacement, and regeneration using the miniature pig as a large animal model.
RESULTS
global transcriptome complexity and gene expression profiles during early morphogenesis of deciduous molars and successional dental lamina
after digital processing of the array data,  <dig>  transcripts  were present in the tooth germs from the three stages, of which  <dig> ,  <dig> , and  <dig>  probes were expressed on e <dig>  e <dig>  and e <dig>  respectively.
of these,  <dig> ,  <dig>  and  <dig> genes were expressed only in tooth germs on e <dig>  e <dig>  and e <dig>  respectively, and  <dig> genes were expressed in all three stages .
large numbers of transcripts were differentially expressed with more than two-fold changes between any two given stages, and  <dig>  genes with annotations were screened for subsequent analysis .
the number of genes with significant differential expression varied between the three stages investigated:  <dig>  between e <dig> and e <dig> ,  <dig> between e <dig> and e <dig> , and  <dig>  between e <dig> and e <dig>  .
venn diagram analysis showed the most changes in gene expression occurred on e <dig> versus e <dig> .
less transcripts were significantly altered between e <dig> and e <dig>   <cit> .
in addition, there were a total of  <dig> genes with significant alterations during all  <dig> stages, with  <dig> genes commonly up-regulated,  <dig> commonly down-regulated, and  <dig> contra-regulated  .
the results suggested the differential gene expression patterns in the different developmental stages of the pig tooth germ were consistent with the transitions of morphogenesis.
during early diphyodont morphogenesis from e <dig> to e <dig>  the dm <dig> underwent  <dig> stages .
the dm <dig> showed dramatic changes in morphology at e <dig>  which reached bell stage with characteristic sdl for secondary permanent tooth, and dm <dig> morphogenesis changed little at e <dig>  without evidence of morphological changes in the sdl.
so, the more changes in transcription between e <dig> and e <dig> implied that more gene variations were involved in dm <dig> morphogenesis from cap stage to bell stage as well as the appearance of sdl the results also showed the genes were predominantly down-regulated on e <dig> versus e40; while, up-regulated genes dominated the morphogenesis of tooth germs on e <dig> versus e <dig>
in summary, the gene expression profiles indicated that spatio-temporal patterns of gene expression were mainly down-regulated; while, both activation and inhibition occurred during diphyodont morphogenesis.
our results provide a molecular basis for studying teeth development, replacement, and regeneration and a better understanding of the dynamic gene regulation of early diphyodont development in the miniature pig.
immunocytochemistry and real-time rt-pcr verification of gene expression
validation of microarray data was achieved using immunocytochemistry and real-time reverse transcription–polymerase chain reaction .
we used immunocytochemistry to detect proteins known to be important for tooth germ cell differentiation .
the results confirmed there were similar changes in expression at the protein level .
nine representative genes were chosen for qrt-pcr, based either on their fold change and/or their potential functions.
the qrt-pcr results were also consistent with the normalized microarray data .
gene ontology analysis of screened genes
gene ontology  analysis of significantly up-regulated differential genes on e <dig> versus e <dig> showed  these genes strongly correlated with multicellular organism development, odontogenesis of dentine-containing teeth, regulation of gene expression, cell adhesion, signal transduction, integrin-mediated signaling pathways, metabolic processes, and apoptosis.
the down-regulated differential genes were mainly involved in muscle organ development .
significantly up-regulated differential genes on e <dig> versus e <dig> correlated strongly with biomineral tissue development, cell redox homeostasis, metabolic processes, cell adhesion, ion transport, multicellular organism development, oxidation-reduction, epithelial cell maturation, apoptosis, integrin-mediated signaling pathways, and the odontogenesis of dentin-containing teeth .
the down-regulated differential genes were mainly involved in metabolic process, signal transduction, ion transport, muscle contraction, blood vessel development, epithelial to mesenchymal transition, inflammatory response, and immune response .
the enriched go analyses of all significant differentially expressed genes between any two stages were associated with  <dig> categories .
there were  <dig> very significant go categories  filtered by p-value and fdr , with the most significant  being “muscle filament sliding”, “cell adhesion”, “chromatin modification”, “signal transduction”, and “mrna processing”, which correlated with down-regulated genes .
we also identified the specific genes that significantly correlated with odontogenesis among the enriched go categories, including odontogenesis of dentine-containing teeth , tooth mineralization , regulation of odontogenesis of dentine-containing teeth , and positive regulation of tooth mineralization  .
furthermore, recent studies suggested dental lamina is a source of odontogenic stem cells, which play an important role in tooth replacement  <cit> .
our results showed the enriched genes linked to the go term molecular function of epithelial development , including wnt and bmp, were confirmed to be involved in tooth replacement in other animals, such as snakes, alligators, and ferrets, which suggests signal molecule conservative in evolution and species  <cit> .
these enriched genes and go groups should be studied further to determine regulation patterns of diphyodont replacement in a large animal model.
pathway analysis of screened genes
pathway analysis was used to determine significant pathways involving the differential genes, according to kegg, biocarta and reatome.
a total of  <dig> significant pathways involved differential genes .
there were  <dig> very significant pathways  , with the most significant  being ecm-receptor interactions, focal adhesion, pathways in cancer, arginine and proline metabolism, and the ppar signaling pathway , some of which were closely correlated with down-regulated genes consistent with down-regulated genes dominating the morphogenesis above mentioned.
for example, the ecm-receptor interaction and focal adhesion pathways are involved in homeostasis.
the extracellular matrix , including the integrin and collagen families, serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function.
the cells and ecm interactions play essential roles in important biological processes including cell adhesion, cell migration, cell survival and apoptosis, cell proliferation, cell differentiation, and regulation of gene expression.
in addition, integrins function as mechanoreceptors and provide a force-transmitting physical link between the ecm and cytoskeleton  <cit> .
taking into account importance of dental lamina on successional odontogenesis, we matched the genes correlated with epithelium determination derived from enriched go categories with  <dig> significant pathways above-mentioned,  <dig> possible pathways were enriched to contribute most likely to diphyodont dentition, including tgf-beta signaling pathway, mapk signaling pathway, cytokine-cytokine receptor interaction and hedgehog signaling pathway .
this prediction is reflected in the subsequent path-net constructed.
series test of cluster  and stc-go analysis of genes
to further narrow the target genes with greatly significant differential expression, we placed  <dig>  genes in sixteen possible model profiles  to enrich the expression tendency of the genes using the series test of cluster  analysis.
we identified seven patterns of gene expression  with significance .
of these, four patterns were most significant , and the enriched go terms most significantly associated with these patterns are shown in figure 4b.
the significant tendencies of gene expression can be grouped into three modes: profiles  <dig>   <dig>   <dig>  and  <dig> contained  <dig>   <dig>   <dig>  and  <dig> genes, respectively, whose expression constantly decreased ; profile  <dig> contained  <dig> genes with constantly increased expression ; profile  <dig> and  <dig> contained  <dig> and  <dig> genes, respectively, characterized by first decreased then slightly increased expression .
the significant enriched go terms from profiles  <dig>   <dig>  and  <dig> are shown in figure 5b.
the lists of assigned genes in each expression profile are included in additional file  <dig>  among these patterns, profile  <dig> had a tendency towards constantly increased gene expression and closely correlated with odontogenesis by stc-go analysis.
the included genes were involved in cell adhesion, odontogenesis of dentine-containing teeth, establishment or maintenance of cell polarity, cellular component movement, epithelial to mesenchymal transition, regulation of epithelial cell migration, mesenchyme development, bmp signaling pathways, tooth mineralization, positive regulation of apoptosis, enamel mineralization, positive regulation of tooth mineralization, organ morphogenesis, etc.
stc analysis suggested that down-regulation patterns were predominant and were required for diphyodont morphogenesis, which further verified the array data described above.
these gene expression profiles indicated that the assigned genes were involved in dynamic activation and inhibition that may play important roles in diphyodont odontogenesis.
further work is necessary to identify gene regulation patterns.
path-net analysis
to further understand the interaction of pathways during diphyodont morphogenesis and screen key pathways with significant role on dental lamina determination, we adopt the strategy to condense information.
the pathways were first filtered by stc analysis to enrich  <dig> significant expression tendencies.
then, an interaction net was built with path-net analysis , according to the graph theory and the interactions among pathways of the kegg database, to enrich the significant pathways very likely related to diphyodont morphogenesis and identify interactions among the significant pathways involving the up-regulated and down-regulated differentially expressed genes .
as shown in figure  <dig>  some differentially expressed genes involved in key pathways during diphyodont morphogenesis were identified, including apoptosis, the mapk signaling pathway, focal adhesion, pathways in cancer, cell cycle, the p <dig> signaling pathway, regulation of actin cytoskeleton, and the tgf-beta signaling pathway, and so on.
moreover, above-predicted  <dig> pathways in pathway analysis  and their interaction shown in figure  <dig> also played central role.
these pathways combined with stc-go analysis further implied that they likely contributed to dental lamina fate and were required in diphyodont odontogenesis.
thus, the pathway profiles involving differentially expressed genes were identified and provided insights into understanding early diphyodont odontogenesis in miniature pigs.
these results, especially pathways related to dental lamina determination, are worthy of further study.
signal-net analysis
gene signal transduction networks  were established to illustrate the inter-gene signaling between the differentially expressed genes.
a total of  <dig> genes were screened as potential targets for diphyodont morphogenesis .
as shown in figure  <dig>  the integrin family may play a key role in early diphyodont morphogenesis and odontogenesis, which are involved in ecm-receptor interactions and focal adhesion pathways.
some studies suggested that integrin not only functions to mediate cell adhesion but also serves as an essential element for proliferation during normal homeostasis  <cit> .
the results implied niche plays a role in tooth development and provided clues for understanding the molecule mechanisms regulating diphyodont morphogenesis.
CONCLUSIONS
here, we characterized the temporal gene expression profiles of diphyodont development at early stages in miniature pigs using microarrays.
we focused on the significant pathways involved and gene-gene interactions in early stage odontogenesis and identified  <dig> genes that very likely correlated with diphyodont development.
we also identified the go terms that significantly correlated with the specific genes involved in the molecular function of odontogenesis, including the odontogenesis of dentine-containing teeth, tooth mineralization, regulation of odontogenesis of dentine-containing teeth, and positive regulation of tooth mineralization.
in our study,  <dig>  differential genes were screened using a series of bioinformatics analyses of diphyodont morphogenesis.
the results indicated that odontogenesis in miniature pigs involves spatio-temporal development of primary teeth and the initiation of secondary dental lamina.
there were stage-special gene expression patterns, in which down-regulated genes were predominant on e <dig> versus e <dig>  while up-regulated genes dominated the morphogenesis of tooth germs on e <dig> versus e <dig>  in brief, fine-tuning activation and inhibition of gene expression plays an important role in tooth morphology and renewal.
these genes form a network and strongly correlate with diphyodont morphogenesis.
taken together, our results provide a molecule basis for studying pigs, which are excellent animal models for studying teeth development and regeneration.
the genes and pathways screened here suggest strong candidates for in-depth studies of the molecular mechanisms of tooth development and diphyodont replacement in miniature pigs.
those results contribute to the understanding of tooth developmental processes in large animal models, which is a fundamental step for investigating the mechanisms of tooth replacement in humans and is applicable to regenerative medicine.

 odontogenesis is driven by many genes encoding signature and signaling molecules, which are regulated by epithelial-mesenchymal interactions mediated by the fine-tuning of conserved signaling pathways including shh, wnt, fgf, tgf-β, bmp, eda, etc.. the current understanding of the molecular mechanisms controlling tooth development has come mostly from studies in mice, which have only one set of non-replaced dentition with a diastema and are obviously different from humans with respect to tooth anatomy and development; therefore, relatively little is known about the mechanisms of tooth replacement in mammals. a desirable model remains a significant obstacle for understanding the mechanisms of tooth replacement. pigs resemble humans in anatomy, physiology, pathophysiology, development, and immune responses, and have been successfully used as an experimental model for craniofacial research. recently, swine have become more popular as a useful pre-clinical model for jaw osteoradionecrosis, jaw bone defects, salivary gland radiation damage, periodontal diseases, craniofacial disorders, temporal mandibular joint fractures, and autoimmune diseases. swine would serve as excellent pre-clinical experiment alternatives for tooth development and regeneration compared with the rodent models widely used. the initiation, eruption time, and sequence of tooth development in miniature pigs are quite similar in humans. in addition, swine have diphyodont dentition, which is an excellent model for studying teeth replacement. the teeth anatomy and deciduous teeth development of miniature pigs have been described previously. to date, there is a lack of gene expression and regulation profiles during odontogenesis in swine and the differences in gene expression profiles between swine, rodents, and human remain largely undefined. recently, the porcine genome project was completed; thus, further analysis of the mechanisms of morphogenetic patterns and diphyodont replacement should be possible using molecular methods in this large animal model. the primary teeth and successional counterparts of wuzhishan miniature pigs have many similarities to humans in size, morphology, number, and form of teeth. we previously determined the patterns of early diphyodont morphogenesis in the wuzhishan miniature pig. the results indicated that the last deciduous molar in the mandible of the miniature pig undergoes cap stage at embryonic day, bell stage at e secretory stage at e and its successional tooth is derived from the lingual successional dental lamina, which is visible at e when its deciduous counterpart is in bell stage, and changes little in morphology at e. the developmental features offer a desirable pattern for understanding diphyodont replacement. moreover, the analysis of the genome sequence of the wuzhishan miniature pig has been published. based on the above studies, we used the wuzhishan miniature pig as a large animal model to investigate the gene expression profiles and functional regulation networks of developing deciduous molars and their replacement counterparts using microarrays. we analyzed the differential gene expression profiles and characterized the gene regulatory networks of morphogenesis and cell differentiation during early diphyodont morphogenesis. we identified the stage-specific genes and pathways with differential regulation. the results provide a molecular base for further research on tooth development, replacement, and regeneration using the miniature pig as a large animal model.  global transcriptome complexity and gene expression profiles during early morphogenesis of deciduous molars and successional dental lamina after digital processing of the array data, transcripts were present in the tooth germs from the three stages, of which,, and probes were expressed on e e and e respectively. of these,, and genes were expressed only in tooth germs on e e and e respectively, and genes were expressed in all three stages. large numbers of transcripts were differentially expressed with more than two-fold changes between any two given stages, and genes with annotations were screened for subsequent analysis. the number of genes with significant differential expression varied between the three stages investigated: between e and e, between e and e, and between e and e. venn diagram analysis showed the most changes in gene expression occurred on e versus e. less transcripts were significantly altered between e and e. in addition, there were a total of genes with significant alterations during all stages, with genes commonly up-regulated, commonly down-regulated, and contra-regulated. the results suggested the differential gene expression patterns in the different developmental stages of the pig tooth germ were consistent with the transitions of morphogenesis. during early diphyodont morphogenesis from e to e the dm underwent stages. the dm showed dramatic changes in morphology at e which reached bell stage with characteristic sdl for secondary permanent tooth, and dm morphogenesis changed little at e without evidence of morphological changes in the sdl. so, the more changes in transcription between e and e implied that more gene variations were involved in dm morphogenesis from cap stage to bell stage as well as the appearance of sdl the results also showed the genes were predominantly down-regulated on e versus e40; while, up-regulated genes dominated the morphogenesis of tooth germs on e versus e in summary, the gene expression profiles indicated that spatio-temporal patterns of gene expression were mainly down-regulated; while, both activation and inhibition occurred during diphyodont morphogenesis. our results provide a molecular basis for studying teeth development, replacement, and regeneration and a better understanding of the dynamic gene regulation of early diphyodont development in the miniature pig. immunocytochemistry and real-time rt-pcr verification of gene expression validation of microarray data was achieved using immunocytochemistry and real-time reverse transcription–polymerase chain reaction. we used immunocytochemistry to detect proteins known to be important for tooth germ cell differentiation. the results confirmed there were similar changes in expression at the protein level. nine representative genes were chosen for qrt-pcr, based either on their fold change and/or their potential functions. the qrt-pcr results were also consistent with the normalized microarray data. gene ontology analysis of screened genes gene ontology analysis of significantly up-regulated differential genes on e versus e showed these genes strongly correlated with multicellular organism development, odontogenesis of dentine-containing teeth, regulation of gene expression, cell adhesion, signal transduction, integrin-mediated signaling pathways, metabolic processes, and apoptosis. the down-regulated differential genes were mainly involved in muscle organ development. significantly up-regulated differential genes on e versus e correlated strongly with biomineral tissue development, cell redox homeostasis, metabolic processes, cell adhesion, ion transport, multicellular organism development, oxidation-reduction, epithelial cell maturation, apoptosis, integrin-mediated signaling pathways, and the odontogenesis of dentin-containing teeth. the down-regulated differential genes were mainly involved in metabolic process, signal transduction, ion transport, muscle contraction, blood vessel development, epithelial to mesenchymal transition, inflammatory response, and immune response. the enriched go analyses of all significant differentially expressed genes between any two stages were associated with categories. there were very significant go categories filtered by p-value and fdr, with the most significant being “muscle filament sliding”, “cell adhesion”, “chromatin modification”, “signal transduction”, and “mrna processing”, which correlated with down-regulated genes. we also identified the specific genes that significantly correlated with odontogenesis among the enriched go categories, including odontogenesis of dentine-containing teeth, tooth mineralization, regulation of odontogenesis of dentine-containing teeth, and positive regulation of tooth mineralization. furthermore, recent studies suggested dental lamina is a source of odontogenic stem cells, which play an important role in tooth replacement. our results showed the enriched genes linked to the go term molecular function of epithelial development, including wnt and bmp, were confirmed to be involved in tooth replacement in other animals, such as snakes, alligators, and ferrets, which suggests signal molecule conservative in evolution and species. these enriched genes and go groups should be studied further to determine regulation patterns of diphyodont replacement in a large animal model. pathway analysis of screened genes pathway analysis was used to determine significant pathways involving the differential genes, according to kegg, biocarta and reatome. a total of significant pathways involved differential genes. there were very significant pathways, with the most significant being ecm-receptor interactions, focal adhesion, pathways in cancer, arginine and proline metabolism, and the ppar signaling pathway, some of which were closely correlated with down-regulated genes consistent with down-regulated genes dominating the morphogenesis above mentioned. for example, the ecm-receptor interaction and focal adhesion pathways are involved in homeostasis. the extracellular matrix, including the integrin and collagen families, serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function. the cells and ecm interactions play essential roles in important biological processes including cell adhesion, cell migration, cell survival and apoptosis, cell proliferation, cell differentiation, and regulation of gene expression. in addition, integrins function as mechanoreceptors and provide a force-transmitting physical link between the ecm and cytoskeleton. taking into account importance of dental lamina on successional odontogenesis, we matched the genes correlated with epithelium determination derived from enriched go categories with significant pathways above-mentioned, possible pathways were enriched to contribute most likely to diphyodont dentition, including tgf-beta signaling pathway, mapk signaling pathway, cytokine-cytokine receptor interaction and hedgehog signaling pathway. this prediction is reflected in the subsequent path-net constructed. series test of cluster and stc-go analysis of genes to further narrow the target genes with greatly significant differential expression, we placed genes in sixteen possible model profiles to enrich the expression tendency of the genes using the series test of cluster analysis. we identified seven patterns of gene expression with significance. of these, four patterns were most significant, and the enriched go terms most significantly associated with these patterns are shown in figure 4b. the significant tendencies of gene expression can be grouped into three modes: profiles and contained and genes, respectively, whose expression constantly decreased; profile contained genes with constantly increased expression; profile and contained and genes, respectively, characterized by first decreased then slightly increased expression. the significant enriched go terms from profiles and are shown in figure 5b. the lists of assigned genes in each expression profile are included in additional file among these patterns, profile had a tendency towards constantly increased gene expression and closely correlated with odontogenesis by stc-go analysis. the included genes were involved in cell adhesion, odontogenesis of dentine-containing teeth, establishment or maintenance of cell polarity, cellular component movement, epithelial to mesenchymal transition, regulation of epithelial cell migration, mesenchyme development, bmp signaling pathways, tooth mineralization, positive regulation of apoptosis, enamel mineralization, positive regulation of tooth mineralization, organ morphogenesis, etc. stc analysis suggested that down-regulation patterns were predominant and were required for diphyodont morphogenesis, which further verified the array data described above. these gene expression profiles indicated that the assigned genes were involved in dynamic activation and inhibition that may play important roles in diphyodont odontogenesis. further work is necessary to identify gene regulation patterns. path-net analysis to further understand the interaction of pathways during diphyodont morphogenesis and screen key pathways with significant role on dental lamina determination, we adopt the strategy to condense information. the pathways were first filtered by stc analysis to enrich significant expression tendencies. then, an interaction net was built with path-net analysis, according to the graph theory and the interactions among pathways of the kegg database, to enrich the significant pathways very likely related to diphyodont morphogenesis and identify interactions among the significant pathways involving the up-regulated and down-regulated differentially expressed genes. as shown in figure  some differentially expressed genes involved in key pathways during diphyodont morphogenesis were identified, including apoptosis, the mapk signaling pathway, focal adhesion, pathways in cancer, cell cycle, the p signaling pathway, regulation of actin cytoskeleton, and the tgf-beta signaling pathway, and so on. moreover, above-predicted pathways in pathway analysis and their interaction shown in figure  also played central role. these pathways combined with stc-go analysis further implied that they likely contributed to dental lamina fate and were required in diphyodont odontogenesis. thus, the pathway profiles involving differentially expressed genes were identified and provided insights into understanding early diphyodont odontogenesis in miniature pigs. these results, especially pathways related to dental lamina determination, are worthy of further study. signal-net analysis gene signal transduction networks were established to illustrate the inter-gene signaling between the differentially expressed genes. a total of genes were screened as potential targets for diphyodont morphogenesis. as shown in figure  the integrin family may play a key role in early diphyodont morphogenesis and odontogenesis, which are involved in ecm-receptor interactions and focal adhesion pathways. some studies suggested that integrin not only functions to mediate cell adhesion but also serves as an essential element for proliferation during normal homeostasis. the results implied niche plays a role in tooth development and provided clues for understanding the molecule mechanisms regulating diphyodont morphogenesis.  here, we characterized the temporal gene expression profiles of diphyodont development at early stages in miniature pigs using microarrays. we focused on the significant pathways involved and gene-gene interactions in early stage odontogenesis and identified genes that very likely correlated with diphyodont development. we also identified the go terms that significantly correlated with the specific genes involved in the molecular function of odontogenesis, including the odontogenesis of dentine-containing teeth, tooth mineralization, regulation of odontogenesis of dentine-containing teeth, and positive regulation of tooth mineralization. in our study, differential genes were screened using a series of bioinformatics analyses of diphyodont morphogenesis. the results indicated that odontogenesis in miniature pigs involves spatio-temporal development of primary teeth and the initiation of secondary dental lamina. there were stage-special gene expression patterns, in which down-regulated genes were predominant on e versus e while up-regulated genes dominated the morphogenesis of tooth germs on e versus e in brief, fine-tuning activation and inhibition of gene expression plays an important role in tooth morphology and renewal. these genes form a network and strongly correlate with diphyodont morphogenesis. taken together, our results provide a molecule basis for studying pigs, which are excellent animal models for studying teeth development and regeneration. the genes and pathways screened here suggest strong candidates for in-depth studies of the molecular mechanisms of tooth development and diphyodont replacement in miniature pigs. those results contribute to the understanding of tooth developmental processes in large animal models, which is a fundamental step for investigating the mechanisms of tooth replacement in humans and is applicable to regenerative medicine. 
the goal of this study is to undertake the survey of differential gene expression profiling and functional network analysis during morphogenesis of diphyodont dentition in miniature pigs. the temporal gene expression profiles during early diphyodont development in miniature pigs were detected with the affymetrix porcine genechip. our study offers a mechanistic framework for understanding dynamic gene regulation of early diphyodont development and provides a molecular basis for studying teeth development, replacement, and regeneration in miniature pigs. our current knowledge of tooth development derives mainly from studies in mice, which have only one set of non-replaced teeth, compared with the diphyodont dentition in humans. the miniature pig is also diphyodont, making it a valuable alternative model for understanding human tooth development and replacement. the gene expression profiles indicated that spatio-temporal down-regulation patterns of gene expression were predominant; while, both dynamic activation and inhibition of pathways occurred during the morphogenesis of diphyodont dentition. the identification of genes related to diphyodont development should lead to a better understanding of morphogenetic patterns and the mechanisms of diphyodont replacement in large animal models and humans. however, little is known about gene expression and function during swine odontogenesis. the gene expression data were further evaluated by anova as well as pathway and stc analyses.
 our current knowledge of tooth development derives mainly from studies in mice, which have only one set of non-replaced teeth, compared with the diphyodont dentition in humans. the miniature pig is also diphyodont, making it a valuable alternative model for understanding human tooth development and replacement. however, little is known about gene expression and function during swine odontogenesis. the goal of this study is to undertake the survey of differential gene expression profiling and functional network analysis during morphogenesis of diphyodont dentition in miniature pigs. the identification of genes related to diphyodont development should lead to a better understanding of morphogenetic patterns and the mechanisms of diphyodont replacement in large animal models and humans.  the temporal gene expression profiles during early diphyodont development in miniature pigs were detected with the affymetrix porcine genechip. the gene expression data were further evaluated by anova as well as pathway and stc analyses. a total of genes were detected with differential expression. several signal pathways and genes were then identified through the construction of pathway and signal networks.  the gene expression profiles indicated that spatio-temporal down-regulation patterns of gene expression were predominant; while, both dynamic activation and inhibition of pathways occurred during the morphogenesis of diphyodont dentition. our study offers a mechanistic framework for understanding dynamic gene regulation of early diphyodont development and provides a molecular basis for studying teeth development, replacement, and regeneration in miniature pigs. gene expression profilediphyodontodontogenesisminiature pig 