Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.
ABSTRACT: Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.
Project description: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 TGF?3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgf?3-induced fusion defect. These findings indicate that IRF6 is involved in TGF?3-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 TGF?3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgf?3-induced fusion defect. These findings indicate that TGF?3 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 TGF?3, which regulates EMT and fusion process during the embryonic palate development.
Project description:Cleft palate, including submucous cleft palate, is among the most common birth defects in humans. While overt cleft palate results from defects in growth or fusion of the developing palatal shelves, submucous cleft palate is characterized by defects in palatal bones. In this report, we show that the Bmpr1a gene, encoding a type I receptor for bone morphogenetic proteins (Bmp), is preferentially expressed in the primary palate and anterior secondary palate during palatal outgrowth. Following palatal fusion, Bmpr1a mRNA expression was upregulated in the condensed mesenchyme progenitors of palatal bone. Tissue-specific inactivation of Bmpr1a in the developing palatal mesenchyme in mice caused reduced cell proliferation in the primary and anterior secondary palate, resulting in partial cleft of the anterior palate at birth. Expression of Msx1 and Fgf10 was downregulated in the anterior palate mesenchyme and expression of Shh was downregulated in the anterior palatal epithelium in the Bmpr1a conditional mutant embryos, indicating that Bmp signaling regulates mesenchymal-epithelial interactions during palatal outgrowth. In addition, formation of the palatal processes of the maxilla was blocked while formation of the palatal processes of the palatine was significantly delayed, resulting in submucous cleft of the hard palate in the mutant mice. Our data indicate that Bmp signaling plays critical roles in the regulation of palatal mesenchyme condensation and osteoblast differentiation during palatal bone formation.
Project description:Cleft palate is a developmental defect resulting from the failure of embryonic palatal shelves to fuse with each other at a critical time. Immediately before and during palatal fusion (E13-E15 in mice), transforming growth factor β3 (TGFβ3) is expressed in the palatal shelf medial edge epithelium (MEE) and plays a pivotal role in palatal fusion. Using Tgfβ3(-/-) mice, which display complete penetrance of the cleft palate phenotype, we tested the hypothesis that intra-amniotic gene transfer could be used to prevent cleft palate formation by restoring palatal midline epithelial function. An adenoviral vector encoding Tgfβ3 was microinjected into the amniotic sacs of mouse embryos at successive developmental stages. Transduced Tgfβ3(-/-) fetuses showed efficient recovery of palatal fusion with mesenchymal confluence following injection at E12.5 (100%), E13.5 (100%), E14.5 (82%), and E15.5 (75%). Viral vectors injected into the amniotic sac transduced the most superficial and transient peridermal cell layer but not underlying basal epithelial cells. TGFβ3 transduction of the peridermdal cell layer was sufficient to induce adhesion, fusion, and disappearance of the palatal shelf MEE in a cell nonautonomous manner. We propose that intra-amniotic gene transfer approaches have therapeutic potential to prevent cleft palate in utero, especially those resulting from palatal midline epithelial dysfunction.
Project description:Secondary palate fusion requires adhesion and epithelial-to-mesenchymal transition (EMT) of the epithelial layers on opposing palatal shelves. This EMT requires transforming growth factor ?3 (TGF?3), and its failure results in cleft palate. Ephrins, and their receptors, the Ephs, are responsible for migration, adhesion, and midline closure events throughout development. Ephrins can also act as signal-transducing receptors in these processes, with the Ephs serving as ligands (termed "reverse" signaling). We found that activation of ephrin reverse signaling in chicken palates induced fusion in the absence of TGF?3, and that PI3K inhibition abrogated this effect. Further, blockage of reverse signaling inhibited TGF?3-induced fusion in the chicken and natural fusion in the mouse. Thus, ephrin reverse signaling is necessary and sufficient to induce palate fusion independent of TGF?3. These data describe both a novel role for ephrins in palate morphogenesis, and a previously unknown mechanism of ephrin signaling.
Project description:Background:Autophagy and apoptosis are involved in embryogenesis. However, little is known about the regulatory mechanism of AMBRA1-mediated autophagy and apoptosis associated with epithelial-mesenchymal transition (EMT) in the development of cleft palate (CP). This study is aimed to elucidate a novel regulatory mechanism by which AMBRA1 regulates autophagy and apoptosis associated with EMT during palatal fusion. Methods:We performed lncRNA and mRNA co-expression profile analysis on embryonic gestation day 14.5 (E14.5) mouse embryos from control (n=3) and all-trans retinoic acid-treated (to induce cleft palate, n=3) C57BL/6J mice. Functional prediction for transcription factor (TF)-target gene relationship, which was obtained using Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses (GO/KEGG) pathway analysis, identified the regulatory "lncRNA-TF-target gene" using the trans model. Results:The trans analysis revealed that some TFs (e.g., LEF1, SMAD4, and FOXD3) regulate lncRNA and gene expression. Finally, we identified a NONMMUT034790.2-LEF1-AMBRA1 trans-regulatory network associated with CP. Our results indicate that AMBRA1 might be a novel epigenetic biomarker in palatogenesis. Conclusions:AMBRA1-mediated autophagy and apoptosis associated with EMT by a NONMMUT034790.2-LEF1-AMBRA1 trans-regulatory network might be an important mechanism underlying dysfunctional palatal fusion.
Project description:Our previous study has demonstrated that knockdown of Grainyhead-like 2(GRHL2) in colorectal cancer (CRC) cells inhibited cell proliferation by targeting ZEB1. This study aimed at researching whether knockdown of GRHL2 promoted CRC progression and metastasis via inducing epithelial-mesenchymal transition (EMT). GRHL2-upregulated SW-620/GRHL2+ and GRHL2-knockdown HCT116/GRHL2-KD, HT29/GRHL2-KD cells and their control cells were generated. The morphological changes after overexpression and knockdown GRHL2 were observed. qRT-PCR, Western blotting, and Immunofluorescence were used to detect EMT markers: E-cadherin, Vimentin, p-catein, ZO-1 and ZEB1 expression. Then, sh-ZEB1 was transfected to GRHL2 knockdown cells to research the relationship between GRHL2 and ZEB1. Transwell and wound healing assays were further performed to detect the impact of GRHL2 on invasion and migration in vitro. CRC cells were injected into mice tail vein to verify the impact of GRHL2 on CRC metastasis. Morphological change of mesenchymal-epithelial transition (MET) could be observed in SW620/GRHL2+ cell. The expression of epithelial markers: E-cadherin, ?-catenin, ZO-1 were up-regulated, while mesenchymal markers: Vimentin was decreased. Meanwhile, opposite EMT morphological change could be observed in HCT116/GRHL2-KD cell, accompanied by reverse change of E-cadherin, ?-catenin, ZO-1, and Vimentin. The expression level of GRHL2 and ZEB1 was found negative in both SW620/GRHL2+ and HCT116/GRHL2-KD cells. Knockdown of ZEB1 by siRNA in HCT116/GRHL2-KD and HT29/GRHL2-KD could upregulate expression of E-cadherin and GRHL2. GRHL2 knockdown also promoted migration, invasion in vitro and CRC metastasis in mice model. In conclusion, GRHL2/ZEB1 axis inhibits CRC progression and metastasis via oppressing EMT.
Project description:Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors, including TGF-? and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail, and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound-healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-? signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel, and suppressed the emergence of CD44(high)CD24(low) cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was downregulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 downregulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-? and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial versus mesenchymal phenotypes and EMT-driven tumor progression.
Project description:The morphogenesis of the mammalian secondary plate is a series of highly dynamic developmental process, including the palate shelves vertical outgrowth, elevation to the horizontal plane and complete fusion in the midline. Extracellular matrix (ECM) proteins not only form the basic infrastructure for palatal mesenchymal cells to adhere via integrins but also interact with cells to regulate their functions such as proliferation and differentiation. ECM remodeling is essential for palatal outgrowth, expansion, elevation, and fusion. Multiple signaling pathways important for palatogenesis such as FGF, TGF ?, BMP, and SHH remodels ECM dynamics. Dysregulation of ECM such as HA synthesis or ECM breakdown enzymes MMPs or ADAMTS causes cleft palate in mouse models. A better understanding of ECM remodeling will contribute to revealing the pathogenesis of cleft palate.
Project description:Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor--Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.
Project description:Cleft palate is among the most common human birth defects. Submucous cleft palate (SMCP) is a subgroup of cleft palate, which may be as common as overt cleft palate. Despite the high frequency of SMCP in humans, only recently have several animal models of SMCP begun to provide insight into the mechanisms by which SMCP develops. In this study, we show that enhanced BMP signaling through constitutively active ACVR1 in palatal epithelium causes submucous cleft palate in mice. In these mutant mice, the fusion of both palatal mesenchyme in hard palate, and muscles in soft palate were hampered by epithelial tissue. During palatal fusion, enhanced SMAD-dependent BMP signaling impaired cell death and altered cell proliferation rate in medial edge epithelium (MEE), and resulted in MEE persistence. At the molecular level, downregulation of ?Np63, which is crucial for normal palatal fusion, in MEE cells was impaired, leading to a reduction in caspase-3 activation. Our study provides a new insight into the etiology of SMCP caused by augmented BMP signaling.