The Wnt1-Cre2 transgene is active in the male germline.

The Wnt1-Cre transgenic mouse line is widely used to express the CRE recombinase in neural crest lineages, but it overexpresses WNT1 itself, which can cause undesired phenotypes. To address this, we and others previously developed a Wnt1-Cre2 line based on the same regulatory elements as Wnt1-Cre but without ectopic Wnt1 expression. However, while Wnt1-Cre2 exhibits normal activity when transmitted from female mice, it exhibits unexpected activity in the male germline. The Wnt1-Cre2 transgene was previously mapped to the E2f1 locus. Several genes in this genomic region exhibit significant expression in spermatogonia or spermatocytes, suggesting that local regulatory elements may be driving ectopic transgene expression. The Wnt1-Cre2 line can therefore be used both as a neural crest specific and a general deleter, and care should be taken when setting up genetic crosses.

IRF6 and TAK1 coordinately promote the activation of HIPK2 to stimulate apoptosis during palate fusion.

Cleft palate is a common craniofacial defect caused by a failure in palate fusion. The palatal shelves migrate toward one another and meet at the embryonic midline, creating a seam. Transforming growth factor-ß3 (TGF-ß3)-induced apoptosis of the medial edge epithelium (MEE), the cells located along the seam, is required for completion of palate fusion. The transcription factor interferon regulatory factor 6 (IRF6) promotes TGF-ß3-induced MEE cell apoptosis by stimulating the degradation of the transcription factor ΔNp63 and promoting the expression of the gene encoding the cyclin-dependent kinase inhibitor p21. Because homeodomain-interacting protein kinase 2 (HIPK2) functions downstream of IRF6 in human cancer cells and is required for ΔNp63 protein degradation in keratinocytes, we investigated whether HIPK2 played a role in IRF6-induced ΔNp63 degradation in palate fusion. HIPK2 was present in the MEE cells of mouse palatal shelves during seam formation in vivo, and ectopic expression of IRF6 in palatal shelves cultured ex vivo stimulated the expression of Hipk2 and the accumulation of phosphorylated HIPK2. Knockdown and ectopic expression experiments in organ culture demonstrated that p21 was required for HIPK2- and IRF6-dependent activation of caspase 3, MEE apoptosis, and palate fusion. Contact between palatal shelves enhanced the phosphorylation of TGF-ß-activated kinase 1 (TAK1), which promoted the phosphorylation of HIPK2 and palate fusion. Our findings demonstrate that HIPK2 promotes seam cell apoptosis and palate fusion downstream of IRF6 and that IRF6 and TAK1 appear to coordinately enhance the abundance and activation of HIPK2 during palate fusion.

Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0.007); and drug resistance to doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a drug-resistant phenotype in vitro.

IRF6 is the mediator of TGFß3 during regulation of the epithelial mesenchymal transition and palatal fusion.

Mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFß3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfß3-induced fusion defect. These findings indicate that IRF6 is involved in TGFß3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFß3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfß3-induced fusion defect. These findings indicate that TGFß3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFß3, which regulates EMT and fusion process during the embryonic palate development.