the human keratin 14 promoter ( k14 ) was used to either conditionally knockout yap or drive yap transgenic over - expression in mouse embryonic ectoderm - derived epithelial tissues . yap conditional knockout ( cko ) embryos were generated through crossing yap mice with k14-cre mice ,   in c57bl/6 background . yap over - expression ( oe ) embryos were generated through breeding col - teto - yap mice with k14-rtta mice , , , ,   in fvb / nj background , in which the constitutively active form of human yap1 protein with the ser127ala mutation was over - expressed upon doxycycline ( dox ) administration . 2  mg / ml of dox in drinking water was given starting from embryonic days 9.5 ( e9.5 ) to e14.5 . all mouse studies were performed in compliance with the protocols approved by the harvard university institutional animal care and use committee . three yap cko embryos at e14.5 , along with the three corresponding three litter controls , were used for tooth germ collection of the yap cko group . similarly , three yap over - expression and three corresponding litter control embryos at e14.5 were used for the yap oe group . the tooth germs were sequentially broken down by 18 , 21 and 25 gauge needles in trizol ( life technologies ) . then total rna was extracted and purified using rnaeasy mini kit ( qiagen ) with on - column dnase ( qiagen ) digestion , and evaluated by the agilent 2100 bioanalyzer ( agilent technologies , ca ) . 1 ) , indicating high quality of the rna samples . to minimize the individual difference from embryos , each rna - seq sample was pooled from the three biologically different e14.5 tooth germs with the same amount of total rna . rna - sequencing was performed on an illumina hiseq 2000 by the biopolymers facility at harvard medical school . paired end 50-nt sequencing strategy was used for all the rna - seq samples to minimize sequencing reading errors . mus 
 musculus genome ( mm9 ) and dnanexus software were used to extract sequencing reads of genes from the rna - seq raw data . rna - seq raw sequencing reads and aligned reads are available through the gene expression omnibus at the accession number of gse65524 . after rna - seq reads were extracted and aligned with the mm9 genome , the relative read ratio of each gene was calculated and further analysis was conducted . we compared the transcriptomic profilings between the yap cko and wild type tooth germs , as well as those between the yap oe and wild type mouse tooth germs . in comparison with the corresponding controls at the 1.5-fold change cutoff , there were 968 down - regulated genes and 979 up - regulated genes in yap cko tooth germs , while there were down - regulated 1289 genes and up - regulated 774 genes in yap over - expression tooth germs ( fig . further analysis revealed many differentially expressed genes between yap cko and yap oe tooth germs . interestingly , we found that some genes in the hox , wnt and laminin families were differentially expressed in the two kinds of the tooth germs . the transcription levels of several hox gene family members were decreased in yap cko tooth germs while their expression levels were increased in yap oe tooth germs ( fig . 3 ) , indicating that these genes could be potential downstream targets of yap in vivo . the regulation of the hoxa1 and hoxc13 gene expression by yap was further validated and functionally analyzed in both mouse and human epithelial cells . similar to the expression changes of the hox genes , several wnt and laminin family members also showed concurrent changes with yap transcripts ( fig . 3 ) , although the members of these two gene families do not have a linear genomic arrangement pattern . the members of the wnt family showing concurrent changes include wnt10b , wnt16 , wnt3 , wnt3a , wnt4 , wnt6 , wnt7a and wnt9b . the transcript levels of the latter six wnt genes were further validated by qpcr in both tooth germs and skin samples . the members of the laminin gene family with concurrent changes consist of lama3 , lama5 , lamb3 and lamc2 . the human keratin 14 promoter ( k14 ) was used to either conditionally knockout yap or drive yap transgenic over - expression in mouse embryonic ectoderm - derived epithelial tissues . yap conditional knockout ( cko ) embryos were generated through crossing yap mice with k14-cre mice ,   in c57bl/6 background . yap over - expression ( oe ) embryos were generated through breeding col - teto - yap mice with k14-rtta mice , , , ,   in fvb / nj background , in which the constitutively active form of human yap1 protein with the ser127ala mutation was over - expressed upon doxycycline ( dox ) administration . 2  mg / ml of dox in drinking water was given starting from embryonic days 9.5 ( e9.5 ) to e14.5 . all mouse studies were performed in compliance with the protocols approved by the harvard university institutional animal care and use committee . three yap cko embryos at e14.5 , along with the three corresponding three litter controls , were used for tooth germ collection of the yap cko group . similarly , three yap over - expression and three corresponding litter control embryos at e14.5 were used for the yap oe group . the tooth germs were sequentially broken down by 18 , 21 and 25 gauge needles in trizol ( life technologies ) . then total rna was extracted and purified using rnaeasy mini kit ( qiagen ) with on - column dnase ( qiagen ) digestion , and evaluated by the agilent 2100 bioanalyzer ( agilent technologies , ca ) . 1 ) , indicating high quality of the rna samples . to minimize the individual difference from embryos , each rna - seq sample was pooled from the three biologically different e14.5 tooth germs with the same amount of total rna . rna - sequencing was performed on an illumina hiseq 2000 by the biopolymers facility at harvard medical school . paired end 50-nt sequencing strategy was used for all the rna - seq samples to minimize sequencing reading errors . mus 
 musculus genome ( mm9 ) and dnanexus software were used to extract sequencing reads of genes from the rna - seq raw data . rna - seq raw sequencing reads and aligned reads are available through the gene expression omnibus at the accession number of gse65524 . after rna - seq reads were extracted and aligned with the mm9 genome , the relative read ratio of each gene was calculated and further analysis was conducted . we compared the transcriptomic profilings between the yap cko and wild type tooth germs , as well as those between the yap oe and wild type mouse tooth germs . in comparison with the corresponding controls at the 1.5-fold change cutoff , there were 968 down - regulated genes and 979 up - regulated genes in yap cko tooth germs , while there were down - regulated 1289 genes and up - regulated 774 genes in yap over - expression tooth germs ( fig . further analysis revealed many differentially expressed genes between yap cko and yap oe tooth germs . interestingly , we found that some genes in the hox , wnt and laminin families were differentially expressed in the two kinds of the tooth germs . the transcription levels of several hox gene family members were decreased in yap cko tooth germs while their expression levels were increased in yap oe tooth germs ( fig . 3 ) , indicating that these genes could be potential downstream targets of yap in vivo . the regulation of the hoxa1 and hoxc13 gene expression by yap was further validated and functionally analyzed in both mouse and human epithelial cells . similar to the expression changes of the hox genes , several wnt and laminin family members also showed concurrent changes with yap transcripts ( fig . 3 ) , although the members of these two gene families do not have a linear genomic arrangement pattern . the members of the wnt family showing concurrent changes include wnt10b , wnt16 , wnt3 , wnt3a , wnt4 , wnt6 , wnt7a and wnt9b . the transcript levels of the latter six wnt genes were further validated by qpcr in both tooth germs and skin samples . the members of the laminin gene family with concurrent changes consist of lama3 , lama5 , lamb3 and lamc2 . in this study , we used rna - seq analysis to compare the transcriptomic profilings of yap cko and yap over - expression mouse tooth germs . we found that some genes in the hox , wnt and laminin families exhibiting concurrent changes with yap transcripts and may be potentially targets of yap . the regulation of hoxa1 and hoxc13 genes by yap was further validated and functionally analyzed in different epithelial cells of mouse tooth germs , skin samples and human keratinocytes . the transcriptomic profiling comparison revealed that hoxa1 , hoxa2 , hoxa3 , hoxa5 , hoxb9 , hoxc13 , hoxc4 , hoxc8 and hoxd1 have differential expression in yap cko and yap oe tooth germs . however , our qpcr results showed that only hoxa1 and hoxc13 had significant concurrent changes in their transcripts . this discrepancy could be due to low abundance of the hox genes in mouse tooth tissues , which may cause high relative ratios . there are also some other gene families showing concurrent transcript changes with yap transcripts , such as the laminin , rho gtpas activating protein and ras - related protein gene family members . further investigation is required to understand the regulation of their expression by yap and the relationships among these gene families .
to identify the downstream target genes of yap , we used rna - seq technology to compare the transcriptomic profilings of yap conditional knockout ( yap cko ) and yap over - expression mouse tooth germs . 
 our results showed that some hox , wnt and laminin family genes had concurrent changes with yap transcripts , indicating that the expression of these genes may be regulated by yap . here , we provide the detailed experimental procedure for the transcriptomic profiling results ( ncbi geo accession number gse65524 ) . 
 the associated study on the regulation of hoxa1 and hoxc13 genes by yap was published in molecular cellular biology in 2015 [ liu et al . , 2015 ] .