Ng endochondral bone development, Sox9 has been demonstrated to regulate chondrocyte hypertrophy by transactivating the Col10a1 gene along with the transcription factor Mef2c [13]. The expression of both Sox9 and Mef2c within the cartilage is required for chondrocyte hypertrophy [14,15]. Cartilage-specific loss of Sox9, independently or in conjunction with Mef2c, results in delayed chondrocyte hypertrophy and shortened long bones, producing a dwarfism phenotype similar to that seen in the Crtl1 knockout mouse [13,15]. Within the heart, the Mef2 family of transcription factors has long been associated with regulation of myocardially-expressed genes, including cardiac alpha-actin and alpha-myosin heavy chain [16?8]. Mef2c knockout mice dieMef2c Regulates Crtl1 Transcriptionaround ED9.5 due to defects in heart looping and chamber formation. 47931-85-1 web Interestingly, Mef2c null embryos fail to form endocardial cushions and have little cardiac jelly, indicating that the establishment of ECM may be affected [19]. In this study we have tested the hypothesis that Mef2c acts as a transcriptional regulator of Crtl1 expression in the developing heart. Immunohistochemical 
and in situ hybridization expression analyses demonstrate that during cardiac development Crtl1 and Mef2c are co-expressed in endocardial and mesenchymal cells. We identified two highly conserved Mef2 binding sites within the 59 upstream sequences of the Crtl1 promoter. Using chromatin immunoprecipitation (ChIP) we show that Mef2c is able to bind to the conserved Mef2 binding sites in vivo during cardiovascular development. Finally, we show that, using an in vitro approach, in fetal mitral valve interstitial cells (VICs), Crtl1 transcription is dependent on Mef2c expression. The data presented here reveal a new role for Mef2c in cardiac development, specifically related to the transcriptional regulation of extracellular matrix protein expression.Crtl1 mRNA probe 15755315 was synthesized and labeled from PCR product using the DIG RNA Sp6/T7 labeling kit (Roche #11175-025-910). The tissues were de-waxed in xylenes and rehydrated through an ethanol series, and then digested with 15 ug/mL of proteinase K. Following proteinase K digestion, the tissues were treated with 0.2 glycine and post-fixed in 4 PFA/ 0.2 gluteraldehyde solution. The tissues were then hybridized with the RNA DIG-labeled probe overnight at 68uC. After thorough washing on the second day, tissues were blocked in 10 sheep serum/1 blocking reagent (Roche #11-093-274-910). The hybridized probe was visualized using Anlotinib web Immuno BCIP/NBT substrate (MP Biochemicals #980771).DNA PrecipitationOligo design. Sense and anti-sense fifty-nucleotide oligos from the Crtl1 promoter sequence containing the potential Mef2 consensus binding sites and flanking sequence were obtained from Operon with a biotin tag on the 59 end of the sense strand (see Table 1). The sense and anti-sense 
oligos were annealed to form double stranded DNA oligos. To disrupt Mef2c binding, mutated oligos were designed where the Mef2 consensus site from 2707 to 2698 was mutated from 59-ctataaataa-39 to 59-ctatagcgaa-39 and the Mef2 consensus site from 2922 to 2913 was mutated from 59ttataaataa-39 to 59-ttatagcgaa-39. Positive and negative controloligos were designed with three Mef2 consensus sites from the muscle creatine kinase gene (known to bind with Mef2) or with mutated sites with random intervening sequence. DNA affinity precipitation assays were performed as described by other.Ng endochondral bone development, Sox9 has been demonstrated to regulate chondrocyte hypertrophy by transactivating the Col10a1 gene along with the transcription factor Mef2c [13]. The expression of both Sox9 and Mef2c within the cartilage is required for chondrocyte hypertrophy [14,15]. Cartilage-specific loss of Sox9, independently or in conjunction with Mef2c, results in delayed chondrocyte hypertrophy and shortened long bones, producing a dwarfism phenotype similar to that seen in the Crtl1 knockout mouse [13,15]. Within the heart, the Mef2 family of transcription factors has long been associated with regulation of myocardially-expressed genes, including cardiac alpha-actin and alpha-myosin heavy chain [16?8]. Mef2c knockout mice dieMef2c Regulates Crtl1 Transcriptionaround ED9.5 due to defects in heart looping and chamber formation. Interestingly, Mef2c null embryos fail to form endocardial cushions and have little cardiac jelly, indicating that the establishment of ECM may be affected [19]. In this study we have tested the hypothesis that Mef2c acts as a transcriptional regulator of Crtl1 expression in the developing heart. Immunohistochemical and in situ hybridization expression analyses demonstrate that during cardiac development Crtl1 and Mef2c are co-expressed in endocardial and mesenchymal cells. We identified two highly conserved Mef2 binding sites within the 59 upstream sequences of the Crtl1 promoter. Using chromatin immunoprecipitation (ChIP) we show that Mef2c is able to bind to the conserved Mef2 binding sites in vivo during cardiovascular development. Finally, we show that, using an in vitro approach, in fetal mitral valve interstitial cells (VICs), Crtl1 transcription is dependent on Mef2c expression. The data presented here reveal a new role for Mef2c in cardiac development, specifically related to the transcriptional regulation of extracellular matrix protein expression.Crtl1 mRNA probe 15755315 was synthesized and labeled from PCR product using the DIG RNA Sp6/T7 labeling kit (Roche #11175-025-910). The tissues were de-waxed in xylenes and rehydrated through an ethanol series, and then digested with 15 ug/mL of proteinase K. Following proteinase K digestion, the tissues were treated with 0.2 glycine and post-fixed in 4 PFA/ 0.2 gluteraldehyde solution. The tissues were then hybridized with the RNA DIG-labeled probe overnight at 68uC. After thorough washing on the second day, tissues were blocked in 10 sheep serum/1 blocking reagent (Roche #11-093-274-910). The hybridized probe was visualized using Immuno BCIP/NBT substrate (MP Biochemicals #980771).DNA PrecipitationOligo design. Sense and anti-sense fifty-nucleotide oligos from the Crtl1 promoter sequence containing the potential Mef2 consensus binding sites and flanking sequence were obtained from Operon with a biotin tag on the 59 end of the sense strand (see Table 1). The sense and anti-sense oligos were annealed to form double stranded DNA oligos. To disrupt Mef2c binding, mutated oligos were designed where the Mef2 consensus site from 2707 to 2698 was mutated from 59-ctataaataa-39 to 59-ctatagcgaa-39 and the Mef2 consensus site from 2922 to 2913 was mutated from 59ttataaataa-39 to 59-ttatagcgaa-39. Positive and negative controloligos were designed with three Mef2 consensus sites from the muscle creatine kinase gene (known to bind with Mef2) or with mutated sites with random intervening sequence. DNA affinity precipitation assays were performed as described by other.