ole of TWIST1 in controlling differentiation of bone and cartilage.Most of these genes were also enriched in the wild-type AVC libraries. Periostin, another ECM protein and a proposed direct target of TWIST1 in osteoblasts, was 
also reduced significantly in the Twist1 null AVC. Other targets of TWIST1 suggested in chick endocardial cushion cells, Cdh11, Mmp2 and Tbx20, showed decreased expression levels in the Twist1 null library but the reductions were not significant. This suggests that TWIST1 may not be a major regulator of these genes in the mouse AVC and more likely acts as a modulator in combination with other AVC-specific transcription factors. A MedChemExpress NVP BGJ398 critical step in EMT is the acquisition of an invasive phenotype by the newly formed mesenchyme cells. Consistent with a role of TWIST1 in controlling the invasion and cell migration phenotype following initial transformation, genes down-regulated in the Twist1 null AVC included kit ligand, the focal adhesion protein Tns1, and the ErbB2/3 ligand Nrg1. During metastatic progression, TWIST1 is thought to prevent the death of newly transformed cells by inhibiting apoptosis. In addition to their role in cell migration, Kitl and Nrg1 are negative regulators of apoptosis. Other regulators of apoptosis down-regulated in the Twist1 null AVC library include Daxx, Ccar1, and the tumor necrosis factor receptor superfamily, member 12A. Interestingly, several noteworthy transcription factors PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22202440 are downregulated in the Twist1 null AVC, suggesting that TWIST1 is acting as part of a transcriptional network. Three members of the SRY family of transcription factors, Sox9, Sox4 and Sox13, are both down-regulated in the Twist1 null library and enriched in the AVC. Zinc finger transcription factors, including Snai1 and two members of Kruppel-like factor family, were also down-regulated. Twist1 and Snai1 are commonly over-expressed in tissues undergoing EMT, have been shown to have genetic interactions in regulating gene expression of ECM proteins, and their combined mutation enhances abnormal cranial suture fusion. Of the 621 genes that were up-regulated in the Twist1 null AVC, GO analysis indicated that many are involved in metabolism and energy production. These included genes critical in the electron transport chain and many of the essential enzymes in glycolysis. 6 Twist1 Targets in Embryonic Heart Valves Twist1 over WT AVC Gene symbol RefSeq accession AVC OFT Atria Ventricles Twist1 2/2 Fold Change P-Value A. Genes down-regulated in Twist1 null AVC Transcriptional regulators Twist1 Snai1 Sox9 Klf4 Sox4 Sox13 Klf7 ECM components Dcn Col3a1 Bgn Tnc Col1a1 Col1a2 Col6a2 NM_007833 NM_009930 NM_007542 NM_011607 NM_007742 NM_007743 NM_146007 60.4 1442.0 49.4 47.7 160.2 234.7 18.4 41.8 2334.3 36.9 523.1 204.2 239.1 8.4 33.6 94.9 0.0 3.6 24.9 126.4 4.9 33.9 108.7 0.0 0.0 19.7 111.1 1.7 4.7 206.9 4.6 4.9 26.2 48.0 2.9 0.097 0.171 0.116 0.127 0.196 0.244 0.199 4.42E-04 8.77E-04 1.37E-03 3.25E-03 3.79E-03 9.51E-03 3.79E-02 NM_011658 NM_011427 NM_011448 NM_010637 NM_009238 NM_011439 NM_033563 918.0 43.5 514.4 32.1 98.8 27.0 13.7 299.1 18.4 376.7 47.8 86.5 29.6 17.3 63.4 2.1 4.1 0.0 0.0 0.0 0.0 55.9 1.4 4.3 0.0 0.0 1.4 0.0 4.6 4.8 113.5 4.5 19.6 3.9 1.5 0.006 0.137 0.263 0.174 0.238 0.180 0.147 8.49E-13 2.92E-03 1.09E-02 1.22E-02 1.32E-02 1.50E-02 4.80E-02 Bone, cartilage and tendon development Papss2 Mgp Bmp2 Bmp1 Cell migration Tns1 Kitl Efnb1 Nrg1 Apoptosis Daxx Tnfrsf12a Ccar1 NM_007829 NM_013749 NM_009810 63.1 12