The above article from FEBS Letters, published online on 1 November 2016 in Wiley Online Library (http://wileyonlinelibrary.com), has been withdrawn by agreement between the Journal Managing Editor, Felix Wieland, and John Wiley & Sons Ltd., on behalf of Federation of European Biochemical Societies. The withdrawal has been agreed following an investigation by the Journal's Editorial Office that found that the data in Figures 4C and 4E appears to have been manipulated.

The molecular regulation of the lung metastasis of pancreatic carcinoma (PCC) is not completely understood. Here, we show that the levels of phosphorylated SMAD3, ZEB1, ZEB2, Snail1, and Snail2 were significantly higher in PCC with lung metastasis than in PCC without lung metastasis. Overexpression of TGFβ1 enhanced the invasiveness of PCC cells, while inhibition of TGFβ1 decreased the invasiveness of PCC cells, which appeared to be conducted by activated TGFβ receptor signaling-induced upregulation of ZEB1, ZEB2, Snail1, and Snail2, suggesting a process of epithelial-mesenchymal transition (EMT). Taken together, our study provides evidence that TGFβ receptor signaling-induced EMT may be responsible for the increased PCC invasiveness to enhance its lung metastasis.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGFβ-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGFβ-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGFβ-interaction. The results point to a network of FGF-, BMP- and TGFβ-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial mesenchymal transition (EMT) is a critical step in cancer metastasis. Some evidences have been provided to verify up-regulation of linc00511 in multiple cancers and oncogenic roles during cancer malignant process. But, the roles of linc00511 on the metastasis of lung cancer are still largely unclear. Our study aims to reveal the functional effects of linc00511 on TGF-β1-induced EMT in lung cancer. Our results showed that knockdown of linc00511 significantly inhibited TGF-β1-induced migration and invasion and down-regulated the mRNA and protein levels of MMP2, MMP9 and MMP12 in TGF-β1 treated SPCA1 and H1975 cells. Also, western blotting results showed that inhibition of linc00511 remarkably suppressed TGF-β1-induced N-cadherin, Vimentin and snail and increased E-cadherin expression in SPCA1 and H1975 cells. Noteworthy, we further found that inhibition of linc00511 could down-regulate TGF-β1-induced ZEB2 mRNA and protein levels by sponging miR-183-5p in SPCA1 and H1975 cells. Taken together, our findings suggested knockdown linc00511 suppressed TGF-β1-induced migration and invasion via inhibiting EMT and MMPs in lung cancer cells.

Actin filament associated protein 1 antisense RNA 1 (named AFAP1-AS1) is a long non-coding RNA and overexpressed in many cancers. This study aimed to identify the role and mechanism of AFAP1-AS1 in lung cancer. The AFAP1-AS1 expression was firstly assessed in 187 paraffin-embedded lung cancer and 36 normal lung epithelial tissues by in situ hybridization. The migration and invasion abilities of AFAP1-AS1 were investigated in lung cancer cells. To uncover the molecular mechanism about AFAP1-AS1 function in lung cancer, we screened proteins that interact with AFAP1-AS1 by RNA pull down and the mass spectrometry analyses. AFAP1-AS1 was highly expressed in lung cancer clinical tissues and its expression was positively correlated with lung cancer patients' poor prognosis. In vivo experiments confirmed that AFAP1-AS1 could promote lung cancer metastasis. AFAP1-AS1 promoted lung cancer cells migration and invasion through interacting with Smad nuclear interacting protein 1 (named SNIP1), which inhibited ubiquitination and degradation of c-Myc protein. Upregulation of c-Myc molecule in turn promoted the expression of ZEB1, ZEB2, and SNAIL gene, which ultimately enhanced epithelial to mesenchymal transition (EMT) and lung cancer metastasis. Understanding the molecular mechanism by which AFAP1-AS1 promotes lung cancer's migration and invasion may provide novel therapeutic targets for lung cancer patients' early diagnosis and therapy.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive type of malignant tumor with a poor prognosis and high mortality. Aberrant activation of hedgehog signaling plays a crucial role in the maintenance and progression of PDA. Here, we report that the dietary bioflavonoid quercetin has therapeutic potential for PDA by targeting sonic hedgehog (SHH) signaling. The effects of quercetin on the proliferation, apoptosis, migration, and invasion of pancreatic cancer cells (PCCs) and tumor growth and metastasis in PDA xenograft mouse models were evaluated. Additionally, SHH signaling activity was determined. Quercetin significantly inhibited PCC proliferation by downregulating c-Myc expression. In addition, quercetin suppressed epithelial-mesenchymal transition (EMT) by reducing TGF-β1 level, which resulted in inhibition of PCC migration and invasion. Moreover, quercetin induced PCC apoptosis through mitochondrial and death receptor pathways. In nude mouse models, PDA growth and metastasis were reduced by quercetin treatment. Mechanically, quercetin exerts its therapeutic effects on PDA by decreasing SHH activity. Interestingly, quercetin-induced SHH inactivation is mainly dependent on Gli2, but not Gli1. Enhance SHH activity by recombinant Shh protein abolished the quercetin-mediated inhibition of PCC proliferation, migration, and invasion. Furthermore, Shh activated TGF-β1/Smad2/3 signaling and promoted EMT by inducing the expression of Zeb2 and Snail1 that eventually resulted in a partial reversal of quercetin-mediated inhibition of PCC migration and invasion. We conclude that quercetin inhibited the growth, migration, and invasion and induced apoptosis of PCCs by antagonizing SHH and TGF-β/Smad signaling pathways. Thus, quercetin may be a potential candidate for PDA treatment.

Keywords: EMT; Pancreatic ductal adenocarcinoma (PDA); Quercetin; Sonic hedgehog signaling; TGF-β1/Smad2/3.

Spontaneously occurring canine oral squamous cell carcinomas (COSCC) are viewed as a useful model for human head and neck squamous cell carcinomas (HNSCC). To date however, the molecular basis of COSCC remains poorly understood. To identify changes pertinent to cancer cells in COSCC, we specifically analyzed tumor cells and matched normal epithelium from clinical formalin-fixed paraffin-embedded specimens using laser-capture-microdissection coupled with RNA-sequencing (RNAseq). Our results identify strong contributions of epithelial-to-mesenchymal transition (EMT), classical tumor-promoting (such as E2F, KRAS, MYC, mTORC1, and TGFB1 signaling) and immune-related pathways in the tumor epithelium of COSCC. Comparative analyses of COSCC with 43 paired tumor/normal HNSCC from The Cancer Genome Atlas revealed a high homology in transcriptional reprogramming, and identified processes associated with cell cycle progression, immune processes, and loss of cellular differentiation as likely central drivers of the disease. Similar to HNSCC, our analyses suggested a ZEB2-driven partial EMT in COSCC and identified selective upregulation of KRT14 and KRT17 in COSCC. Beyond homology in transcriptional signatures, we also found therapeutic vulnerabilities strongly conserved between the species: these included increased expression of PD-L1 and CTLA-4, coinciding with EMT and revealing the potential for immune checkpoint therapies, and overexpression of CDK4/6 that sensitized COSCC to treatment with palbociclib. In summary, our data significantly extend the current knowledge of molecular aberrations in COSCC and underline the potential of spontaneous COSCC as a model for HNSCC to interrogate therapeutic vulnerabilities and support translation of novel therapies from bench to bedside.

Keywords: CDK4/CDK6; Comparative oncology; FFPE tissue; Laser-capture microdissection; RNA sequencing; palbociclib.

Epithelial-to-mesenchymal transition (EMT) has an active role in the malignant progression of epithelial tumor cells. The aim of the study was to identify the existence of the EMT mechanism in brain metastasis. Tumors from 29 patients with brain metastases were assessed in terms of the immunoexpression of EMT-related factors including Slug, ZEB1, ZEB2, and E-cadherin in tumor cells and the surrounding mesenchymal stromal cells. The results were compared between primary tumors and their matched metastatic brain tumors. Analysis of tumor cell expression showed that Slug, ZEB1, or ZEB2 expression was found in more than 10% of the neoplastic cells in the metastatic lesions of 17 cases (59%) and in the primary lesions of 7 cases (24%, P=0.02). The expression level of ZEB2 was negatively correlated with that of E-cadherin (P=0.05). There were no differences in the tumoral expression levels of Slug, ZEB1, or ZEB2 among the primary organs. Analysis of stromal cell expression revealed a global increase in ZEB1 and ZEB2 expression levels with metastases (P<0.0001). Quantitative analysis confirmed that messenger RNA expression of ZEB1 and ZEB2 was elevated in metastatic lesions. The increased expression of EMT-related factors in brain metastasis was found not only in tumor cells, but also in tumor-associated stromal cells. Our results suggest that EMT-related factors play a role as a facilitator of brain metastasis.

Extra-central nervous system metastasis of gliomas is extremely rare, and the biological mechanism underlying it remains poorly understood. Epithelial-to-mesenchymal transition (EMT) has received attention as one of the important processes of cancer metastasis. Here we describe the case of a 32-year-old man with cutaneous metastasis of high-grade glioma, together with the analysis of EMT-related molecules. Our patient presented with a high-grade glioma in the right frontal lobe. Cutaneous metastasis under the surgical scar developed 17 months after complete resection of the intracranial tumor. Histopathology of both the original and metastatic tumors revealed hypercellularity; the tumors predominantly comprised glial tumor cells with poor cellular processes. Immunohistochemical analysis demonstrated intense expression of nestin, focal expression of glial fibrillary acid protein, and absence of expression of oligodendrocyte transcription factor 2, endothelial membrane antigen, or neurofilament. Genetic analyses could not provide definitive diagnostic information of glioma subtypes. Immunohistochemical analysis for EMT-related biomarkers demonstrated increased Twist, zinc finger E-box-binding homeobox 2 (ZEB2), matrix metalloproteinase 2 (MMP2), and MMP9 expressions in tumor cells of the metastatic lesion compared with those of the primary lesion. Slug, E-cadherin, and N-cadherin expression were absent in both primary and metastatic lesions; however, ZEB1 expression was present in both. Our results suggest that Twist, ZEB2, MMP2, and MMP9 facilitate cutaneous metastasis of gliomas.

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor that is frequently mutated in human cancers. Our previous findings have indicated that SPOP is mutated and functions as a novel tumor suppressor in hepatoblastoma (HB). However, the biological roles and clinical significance of this SPOP in hepatocellular carcinoma (HCC) remain unknown. In this study, we found that the expression level of SPOP was downregulated in HCC primary tumors by quantitative real-time PCR and the protein level of SPOP was also reduced in 72 pairs of HCC tissue microarrays by immunohistochemical analyses. Moreover, SPOP expression was observed to negatively correlate with the tumor grade and intrahepatic metastasis of HCC patients. Furthermore, we revealed that SPOP not only inhibits cell proliferation but also inhibits the motility of liver cancer cells. Finally, we discovered that one of the mechanisms through which SPOP inhibits liver cancer cell migration involves the disruption of ZEB2 expression and the associated epithelial-mesenchymal transition program. Together, our findings emphasize the critical role of SPOP in the regulation of proliferation and migration in liver cancer.

Many chromosomal deletions encompassing the 2q23.1 region have been described ranging from small deletions of 38 kb up to >19 Mb. Most phenotypic features of the 2q23.1 deletion syndrome are due to a MBD5 gene loss independent of the size of the deletion. Here, we describe a male patient harboring a novel interstitial deletion encompassing the 2q22.3 q23.3 chromosomal region. Array-CGH revealed a 7.1 Mb deletion causing haploinsufficiency of several genes including MBD5, ACVR2, KIF5C, and EPC2. This patient presents with additional findings to those already described in individuals who have deletions of MBD5 including toes absence of halluces, pure red cell aplasia, and intestinal aganglionosis. Interestingly, in the deleted region there are previously identified regulatory sequences which are located upstream to ZEB2, which is associated with Hirschsprung disease (HSCR). Several genes have been associated with pure red cell aplasia, but to our knowledge, this is the first time that 2q deletion is associated with this phenotype. These additional findings should be added to the list of manifestations associated with 2q deletion, and provide support for the hypothesis that this individual has a true contiguous gene deletion syndrome.

ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms.

Tubulointerstitial fibrosis (TIF) is the main pathologic feature of end-stage renal disease. Epithelial-mesenchymal transition (EMT) of proximal tubular cells (PTCs) is one of the most significant features of TIF. MicroRNAs play critical roles during EMT in TIF. However, whether miRNAs can be used as therapeutic targets in TIF therapy remains undetermined. We found that miR-30e, a member of the miR-30 family, is deregulated in TGF-β1-induced PTCs, TIF mice and human fibrotic kidney tissues. Moreover, transcription factors that induce EMT, such as snail, slug, and Zeb2, were direct targets of miR-30e. Using a cell-based miR-30e promoter luciferase reporter system, Schisandrin B (Sch B) was selected for the enhancement of miR-30e transcriptional activity. Our results indicate that Sch B can decrease the expression of snail, slug, and Zeb2, thereby attenuating the EMT of PTCs during TIF by upregulating miR-30e, both in vivo and in vitro. This study shows that miR-30e can serve as a therapeutic target in the treatment of patients with TIF and that Sch B may potentially be used in therapy against renal fibrosis.

BRAF inhibitors (BRAFi) and the combination therapy of BRAF and MEK inhibitors (MEKi) were recently approved for therapy of metastatic melanomas harbouring the oncogenic BRAFV600 mutation. Although these therapies have shown pronounced therapeutic efficacy, the limited durability of the response indicates an acquired drug resistance that still remains mechanistically poorly understood at the molecular level. We conducted transcriptome gene profiling in BRAFi-treated melanoma cells and identified that Mer tyrosine kinase (MerTK) is specifically upregulated. MerTK overexpression was demonstrated not only in melanomas resistant to BRAFi monotherapy (5 out of 10 samples from melanoma patients) but also in melanoma resistant to BRAFi+MEKi (1 out of 3), although MEKi alone does not affect MerTK. Mechanistically, BRAFi-induced activation of Zeb2 stimulates MerTK in BRAFV600 melanoma through mTORC1-triggered activation of autophagy. Co-targeting MerTK and BRAFV600 significantly reduced tumour burden in xenografted mice, which was pheno-copied by co-inhibition of autophagy and mutant BRAFV600.

Maintenance and differentiation of progenitor cells in the developing enteric nervous system are controlled by molecules such as the signaling protein endothelin 3 (EDN3), its receptor (the endothelin receptor type B [EDNRB]), and the transcription factors SRY-box 10 (SOX10) and zinc finger E-box binding homeobox 2 (ZEB2). We used enteric progenitor cell (EPC) cultures and mice to study the roles of these proteins in enteric neurogenesis and their cross regulation.

We performed studies in mice with a Zeb2 loss-of-function mutation (Zeb2Δ) and mice carrying a spontaneous recessive mutation that prevents conversion of EDN3 to its active form (Edn3ls). EPC cultures issued from embryos that expressed only wild-type Zeb2 (Zeb2+/+ EPCs) or were heterozygous for the mutation (Zeb2Δ/+ EPCs) were exposed to EDN3; we analyzed the effects on cell differentiation using immunocytochemistry. In parallel, Edn3ls mice were crossed with Zeb2Δ/+mice; intestinal tissues were collected from embryos for immunohistochemical analyses. We investigated regulation of the EDNRB gene in transactivation and chromatin immunoprecipitation assays; results were validated in functional rescue experiments using transgenes expression in EPCs from retroviral vectors.

Zeb2Δ/+ EPCs had increased neuronal differentiation compared to Zeb2+/+ cells. When exposed to EDN3, Zeb2+/+ EPCs continued expression of ZEB2 but did not undergo any neuronal differentiation. Incubation of Zeb2Δ/+ EPCs with EDN3, on the other hand, resulted in only partial inhibition of neuronal differentiation. This indicated that 2 copies of Zeb2 are required for EDN3 to prevent neuronal differentiation. Mice with combined mutations in Zeb2 and Edn3 (double mutants) had more severe enteric anomalies and increased neuronal differentiation compared to mice with mutations in either gene alone. The transcription factors SOX10 and ZEB2 directly activated the EDNRB promoter. Overexpression of EDNRB in Zeb2Δ/+ EPCs restored inhibition of neuronal differentiation, similar to incubation of Zeb2+/+ EPCs with EDN3.

In studies of cultured EPCs and mice, we found that control of differentiation of mouse enteric nervous system progenitor cells by EDN3 requires regulation of Ednrb expression by SOX10 and ZEB2.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Biomarkers of the therapeutic efficacy of ICIs have been extensively investigated. In this study, we aimed to analyze whether molecular phenotypes of circulating tumor cells (CTCs) are associated with treatment responses and clinical outcomes in patients with R/M HNSCC treated with nivolumab. Peripheral blood samples were collected before treatment initiation and after four infusions of nivolumab. CTCs isolated by depletion of CD45-positive cells were analyzed to determine the expression of EPCAM, MET, KRT19, and EGFR using real-time quantitative polymerase chain reaction. CTC-positive samples were analyzed to determine the expression of PIK3CA, CCND1, SNAI1, VIM, ZEB2, CD44, NANOG, ALDH1A1, CD47, CD274, and PDCD1LG2. Of 30 patients treated with nivolumab, 28 (93.3%) were positive for CTCs. In 20 CTC-positive patients, molecular alterations in CTCs before and after nivolumab treatment were investigated. Patients with MET-positive CTCs had significantly shorter overall survival than those with MET-negative CTCs (p = 0.027). The expression level of CCND1 in CTCs of disease-controlled patients was significantly higher than that of disease-progressed patients (p = 0.034). In disease-controlled patients, the expression level of CCND1 in CTCs significantly decreased after nivolumab treatment (p = 0.043). The NANOG expression in CTCs was significantly increased in disease-controlled patients after nivolumab treatment (p = 0.036). Our findings suggest that the molecular profiling of CTCs is a promising tool to predict the treatment efficacy of nivolumab.

Snail and Zeb transcription factors induce epithelial-to-mesenchymal transition (EMT) in embryonic and adult tissues by direct repression of E-cadherin transcription. The repression of E-cadherin transcription by the EMT inducers Snail1 and Zeb2 plays a fundamental role in defining embryonic territories in the mouse, as E-cadherin needs to be downregulated in the primitive streak and in the epiblast, concomitant with the formation of mesendodermal precursors and the neural plate, respectively. Here, we show that in the chick embryo, E-cadherin is weakly expressed in the epiblast at pre-primitive streak stages where it is substituted for by P-cadherin We also show that Snail2 and Zeb2 repress P-cadherin transcription in the primitive streak and the neural plate, respectively. This indicates that E- and P-cadherin expression patterns evolved differently between chick and mouse. As such, the Snail1/E-cadherin axis described in the early mouse embryo corresponds to Snail2/P-cadherin in the chick, but both Snail factors and Zeb2 fulfil a similar role in chick and mouse in directly repressing ectodermal cadherin genes to contribute to the delamination of mesendodermal precursors at gastrulation and the proper specification of the neural ectoderm during neural induction.

Natural killer (NK)-cell development and maturation is a well-organized process. The steroid receptor coactivator 3 (SRC-3) is a regulator of the hematopoietic and immune systems; however, its role in NK cells is poorly understood. Here, SRC-3 displayed increased nuclear translocation in NK cells during terminal differentiation and upon inflammatory cytokine stimulation. Targeted deletion of SRC-3 altered normal NK-cell distribution and compromised NK-cell maturation. SRC-3 deficiency led to significantly impaired NK-cell functions, especially their antitumor activity. The expression of several critical T-bet target genes, including Zeb2, Prdm1, and S1pr5, but not T-bet itself, was markedly decreased in NK cells in the absence of SRC-3. There was a physiologic interaction between SRC-3 and T-bet proteins, where SRC-3 was recruited by T-bet to regulate the transcription of the aforementioned genes. Collectively, our findings unmask a previously unrecognized role of SRC-3 as a coactivator of T-bet in NK-cell biology and indicate that targeting SRC-3 may be a promising strategy to increase the tumor surveillance function of NK cells.

Background: Upregulation of circHIPK3 has been observed in several kinds of malignancies. However, the mechanisms of circHIPK3 in HCC metastases remains unclear. We investigated the role and the mechanisms of circHIPK3 in the development of HCC.

Methods: HCC tissues and paired adjacent non-tumor tissues of surgical patients were used to evaluate circHIPK3 expression. A series of biological experiments had been taken to evaluate the pro-metastatic ability of circHIPK3 during HCC development in vitro and in vivo. The potential mechanisms of circHIPK3 in HCC development were identified by RT-qPCR, Western blot, RIP, and luciferase reporter assays.

Results: CircHIPK3 expression is significantly upregulated during HCC development. Overexpression of circHIPK3 promotes cell migration, invasion, and metastases in vitro and in vivo. CircHIPK3 promoted HCC metastases by sponging miR-338-3p to regulate EMT-associated proteins E-cadherin, vimentin, and ZEB2 expression.

Conclusion: CircHIPK3 plays a regulatory role in metastatic HCC by sponging miR-338-3p to induce ZEB2 expression, thus promoting EMT procession.

Keywords: CircHIPK3; EMT; HCC; ZEB2; miR-338-3p.

ZEB2 is a transcription factor with established roles in neurogenesis but no defined function in postnatal brain despite extensive neuronal expression in telencephalic structures. Multiple, incompletely annotated transcripts derive from the Zeb2 locus; the purpose of the present study was to structurally characterize rat brain Zeb2 transcripts with respect to 3' untranslated (UTR) sequence in order to understand Zeb2 transcript regulation including possible interactions with regulatory molecules such as neuronal miRNAs. We cloned a 5054-nucleotide Zeb2 3' UTR that is included in the most abundant Zeb2 transcript in neonatal rat brain. Unique features of the distal 3' UTR region included a number of brain-specific miRNA target sites; a highly conserved miR-9 target site at 3' UTR position 4097 was selected for functional verification in transfection experiments. Parallel analysis of Zeb2 transcript, ZEB2 protein and miR-9 levels across postnatal brain cortical development revealed a significant accumulation of ZEB2 protein levels only between postnatal days P2 and P5 that was associated with an acute loss of postnatal miR-9 expression in cortex. These studies demonstrate novel features of Zeb2 gene expression in postnatal rat brain and highlight the importance of full transcript annotation for identifying the complement of potential transcript-interacting regulators.

Primary glomerulocystic kidney disease is a special form of renal cystic disorder characterized by Bowman's space dilatation in the absence of tubular cysts. ZEB2 is a SMAD-interacting transcription factor involved in Mowat-Wilson syndrome, a congenital disorder with an increased risk for kidney anomalies. Here we show that deletion of Zeb2 in mesenchyme-derived nephrons with either Pax2-cre or Six2-cre causes primary glomerulocystic kidney disease without tubular cysts in mice. Glomerulotubular junction analysis revealed many atubular glomeruli in the kidneys of Zeb2 knockout mice, which explains the presence of glomerular cysts in the absence of tubular dilatation. Gene expression analysis showed decreased expression of early proximal tubular markers in the kidneys of Zeb2 knockout mice preceding glomerular cyst formation, suggesting that defects in proximal tubule development during early nephrogenesis contribute to the formation of congenital atubular glomeruli. At the molecular level, Zeb2 deletion caused aberrant expression of Pkd1, Hnf1β, and Glis3, three genes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons and is required for proximal tubule development and glomerulotubular junction formation. Our findings also suggest that ZEB2 might be a novel disease gene in patients with primary glomerular cystic disease.