FOXJ1 Expression Research Articles

Prdm16 is required for the maintenance of neural stem cells in the postnatal forebrain and their differentiation into ependymal cells.

We and others showed previously that PR domain-containing 16 (Prdm16) is a transcriptional regulator required for stem cell function in multiple fetal and neonatal tissues, including the nervous system. However, Prdm16 germline knockout mice died neonatally, preventing us from testing whether Prdm16 is also required for adult stem cell function. Here we demonstrate that Prdm16 is required for neural stem cell maintenance and neurogenesis in the adult lateral ventricle subventricular zone and dentate gyrus. We also discovered that Prdm16 is required for the formation of ciliated ependymal cells in the lateral ventricle. Conditional Prdm16 deletion during fetal development using Nestin-Cre prevented the formation of ependymal cells, disrupting cerebrospinal fluid flow and causing hydrocephalus. Postnatal Prdm16 deletion using Nestin-CreERT2 did not cause hydrocephalus or prevent the formation of ciliated ependymal cells but caused defects in their differentiation. Prdm16 was required in neural stem/progenitor cells for the expression of Foxj1, a transcription factor that promotes ependymal cell differentiation. These studies show that Prdm16 is required for adult neural stem cell maintenance and neurogenesis as well as the formation of ependymal cells.

EPND-01. THE ACTIVE REGULATORY LANDSCAPE OF EPENDYMAL TUMORS

AbstractGenomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumour entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas commonly resist chemotherapy and lack available targeted agents for clinical trial development. In the case of genomically balanced ependymomas, and those driven by fusion oncoproteins, we hypothesized that the chromatin landscapes could uncover oncogenes that would inform actionable targets for therapy and reveal specific transcriptional circuitries to identify the molecular origins of the disease. To this end, we mapped active chromatin landscapes in 42 primary ependymomas in discovery and validation cohorts using H3K27 acetylation ChIP-seq. Enhancer regions revealed novel oncogenes, molecular targets, and pathways, which when subjected to small molecule inhibitor or shRNA treatment, increased survival and halted proliferation in mouse and neurosphere patient-derived models of ependymomas. Reconstruction of enhancer networks permitted the identification of core transcription factors (TFs) that establish ependymoma cell state, and lineage-specifying TFs that dictate molecular subgroup identity. Lineage-associated TFs point to distinct spatio-temporal origins of ependymoma subgroups such as FOXJ1 TF activity and expression observed preferentially in subsets of hindbrain ependymomas. To translate our results into a potential clinical paradigm, we demonstrate cancer dependencies on super enhancer associated genes and lineage TFs, and establish the utility of chromatin landscape analysis to predict novel targets for cancer therapy.

Expression of FOXJ1 and ITGB4 is Activated upon KLH and LTA Stimulation in the DT40 Cell Line

Expression of FOXJ1 and ITGB4 is Activated upon KLH and LTA Stimulation in the DT40 Cell Line

Distinct patterns of primary and motile cilia in Rathke's cleft cysts and craniopharyngioma subtypes

Cilia are highly conserved organelles, which serve critical roles in development and physiology. Motile cilia are expressed in a limited range of tissues, where they principally regulate local extracellular fluid dynamics. In contrast, primary cilia are expressed by many vertebrate cell types during interphase, and are intimately involved in the cell cycle and signal transduction. Notably, primary cilia are essential for vertebrate hedgehog pathway activity. Improved detection of motile cilia may assist in the diagnosis of some pathologic entities such as Rathke's cleft cysts, whereas characterizing primary cilia in neoplastic tissues may implicate cilia-dependent signaling pathways as critical for tumorigenesis. We show that immunohistochemistry for the nuclear transcription factor FOXJ1, a master regulator of motile ciliogenesis, robustly labels the motile ciliated epithelium of Rathke's cleft cysts. FOXJ1 expression discriminates Rathke's cleft cysts from entities in the sellar/suprasellar region with overlapping histologic features such as craniopharyngiomas. Co-immunohistochemistry for FOXJ1 and markers that highlight motile cilia such as acetylated tubulin (TUBA4A) and the small GTPase ARL13B further enhance the ability to identify diagnostic epithelial cells. In addition to highlighting motile cilia, ARL13B immunohistochemistry also robustly highlights primary cilia in formalin-fixed paraffin-embedded sections. Primary cilia are present throughout the neoplastic epithelium of adamantinomatous craniopharyngioma, but are limited to basally oriented cells near the fibrovascular stroma in papillary craniopharyngioma. Consistent with this differing pattern of primary ciliation, adamantinomatous craniopharyngiomas express significantly higher levels of SHH, and downstream targets such as PTCH1 and GLI2, compared with papillary craniopharyngiomas. In conclusion, motile ciliated epithelium can be readily identified using immunohistochemistry for FOXJ1, TUBA4A, and ARL13B, facilitating the diagnosis of Rathke's cleft cysts. Primary cilia can be identified by ARL13B immunohistochemistry in routine pathology specimens. The widespread presence of primary cilia in adamantinomatous craniopharyngioma implicates cilia-dependent hedgehog signaling in the pathogenesis of adamantinomatous craniopharyngioma.

Expression and localization of forkhead box protein FOXJ1 in S100β-positive multiciliated cells of the rat pituitary

S100β-positive cells exist in the marginal cell layer (MCL) of the adenohypophysis and follicle structure in the parenchyma of anterior lobe (ALFS) in pituitary. They have multiple functions as phagocytes or cells that regulate hormone secretion. Majority of S100β-positive cells in the adenohypophysis express sex determining region Y-box 2 protein (SOX2), a stem cell marker; therefore, S100β/SOX2 double positive cells are also considered as one type of stem/progenitor cells. MCL and ALFS are consisting of morphologically two types of cells, i.e., multiciliated cells and non-ciliated cells. However, the relationship between the S100β-positive cells and multiciliated cells in the pituitary is largely unknown. In the present study, we first immunohistochemically verified the feature of multiciliated cells in MCL and ALFS. We then examined the expression patterns of FOXJ1, an essential expression factor for multiciliated cell-differentiation, and SOX2 in the S100β-positive multiciliated cells by in situ hybridization and immunohistochemistry. We identified anew the S100β/SOX2/FOXJ1 triple positive multiciliated cells, and revealed that they were dispersed throughout the MCL and ALFS. These results indicate that the MCL and ALFS are consisting of morphologically and functionally distinct two types of cells, i.e., S100β/SOX2 double positive non-ciliated cells and S100β/SOX2/FOXJ1 triple positive multiciliated cells.

Dystroglycan Suppresses Notch to Regulate Stem Cell Niche Structure and Function in the Developing Postnatal Subventricular Zone

While the extracellular matrix (ECM) is known to regulate neural stem cell quiescence in the adult subventricular zone (SVZ), the function of ECM in the developing SVZ remains unknown. Here, we report that the ECM receptor dystroglycan regulates a unique developmental restructuring of ECM in the early postnatal SVZ. Dystroglycan is furthermore required for ependymal cell differentiation and assembly of niche pinwheel structures, at least in part by suppressing Notch activation in radial glial cells, which leads to the increased expression of MCI, Myb, and FoxJ1, transcriptional regulators necessary for acquisition of the multiciliated phenotype. Dystroglycan also regulates perinatal radial glial cell proliferation and transition into intermediate gliogenic progenitors, such that either acute or constitutive loss of function in dystroglycan results in increased oligodendrogenesis. These findings reveal a role for dystroglycan in orchestrating both the assembly and function of the SVZ neural stem cell niche.

Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner

BackgroundThe use of electronic (e)-cigarettes is increasing rapidly, but their lung health effects are not established. Clinical studies examining the potential long-term impact of e-cigarette use on lung health will...

Cilia are required for asymmetric nodal induction in the sea urchin embryo

BackgroundLeft-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet.ResultsHere we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.ConclusionsBased on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0128-7) contains supplementary material, which is available to authorized users.

The Histologic Classifications of Lung Adenocarcinomas Are Discriminable by Unique Lineage Backgrounds

ObjectivesLung adenocarcinomas are a heterogeneous set of diseases with distinct genetic and histologic characteristics. Besides the discovery of oncogenic mutations and introduction of the histologic classifications (2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society and 2015 WHO), increasing evidence has linked this intertumor heterogeneity to the lung lineage–specific pathways and lineage genes. Therefore, in this study, we assessed the gene expression of identified lung lineage genes to study their role in distinguishing lung adenocarcinoma diversities. MethodsA total of 278 surgically resected lung adenocarcinomas were included. Each case was evaluated for genetic mutations and histologic classification. Lineage genes associated with respiratory tract differentiation (NK2 homeobox 1 gene [NKX2-1], GATA protein binding 6 gene [GATA6], foxhead box J1 gene [FOXJ1], and SAM pointed domain containing ETS transcription factor gene [SPDEF]) and stem/basal-like status (inhibitor of DNA binding 2, HLH protein gene [ID2], POU class 5 homeobox 1 gene [POU5F1], SRY-box 2 gene [SOX2], and v-myc avian myelocytomatosis viral oncogene homolog gene [MYC]) were selected. mRNA expression of these genes in each tumor sample was assessed by quantitative real-time polymerase chain reaction and normalized to paired normal lung tissue. ResultsDistinct lineage gene expressions were found on the basis of genetic and histologic diversities. Expression of NKX2-1, GATA6, FOXJ1, and POU5F1 exhibited a significant linear relationship across histologic subgroups that was independent of genetic mutation status. Expression levels of NKX2-1 and POU5F1 were also associated with EGFR mutation status, independent of histologic subtypes. Further analysis revealed that the overexpression of SPDEF defined longer relapse-free survivals, especially in stage I disease. ConclusionsFor the first time, we showed the unique lineage backgrounds of different histologic subtypes and oncogenic mutations. Assessing this added parameter might be beneficial in discriminating intertumor heterogeneity, advancing target exploration, developing theranostic/prognostic biomarkers, and designing clinical trials.

Role of IFN-γ, IL-13, and IL-17 on mucociliary differentiation of nasal epithelial cells in chronic rhinosinusitis with nasal polyps.

Mucociliary dysfunction is a prominent pathophysiological feature of chronic rhinosinusitis with nasal polyps (CRSwNP); however, the precise mechanisms underlying mucociliary dysfunction are still unclear. The aim of this study was therefore to evaluate the effects of IFN-γ, IL-13, and IL-17 on human nasal mucociliary differentiation and ciliary beat frequency (CBF) in patients with CRSwNP. Human nasal epithelial cells from tissue of patients with CRSwNP and control subjects were established as air-liquid interface (ALI) primary cultures. Confluent cultures were incubated with10 ng/mL each of IFN-γ, IL-13, or IL-17 for 14 days and assessed for expression of specific morphological markers and factors associated with mucociliary differentiation, the percentage of ciliated and goblet cells, and CBF. In comparison with control subjects, percentage of ciliated cells and CBF were decreased; while percentage of goblet cells, FOXJ1, and MUC5AC mRNA expression were increased in nasal polyp-derived epithelial cultures. Treatment with IFN-γ and IL-13 significantly decreased the expression of β-tubulin IV (specific cilia marker), ciliated cell number, and expression of FOXJ1 and DNAI2, in epithelial cultures derived from both CRSwNP patients and control subjects. Furthermore, while both IFN-γ and IL-13 treatment significantly decreased the CBF of cells from both CRSwNP patients and control subjects, IL-13 additionally significantly increased goblet cell number and the expression of MUC5AC and CLCA1, in these cultures. IL-17 treatment did not significantly affect ciliated or goblet cell differentiation, CBF, nor MUC5AC and CLCA1 expression, but increased both MUC5B mRNA and protein expression in these cultures. The demonstration that IFN-γ and IL-13 both significantly reduce ciliated cell differentiation and CBF in CRSwNP patients, and IL-13 additionally induces significant goblet cell hyperplasia and MUC5AC mucin expression, as well as IL-17 significantly increases MUC5B mucin expression, suggests that these inflammatory cytokines may be potential therapeutic targets in the management of CRSwNP.

Decreased FOXJ1 expression and its ciliogenesis programme in aggressive ependymoma and choroid plexus tumours.

Well-differentiated human cancers share transcriptional programmes with the normal tissue counterparts from which they arise. These programmes broadly influence cell behaviour and function and are integral modulators of malignancy. Here, we show that the master regulator of motile ciliogenesis, FOXJ1, is highly expressed in cells along the ventricular surface of the human brain. Strong expression is present in cells of the ependyma and the choroid plexus as well as in a subset of cells residing in the subventricular zone. Expression of FOXJ1 and its transcriptional programme is maintained in many well-differentiated human tumours that arise along the ventricle, including low-grade ependymal tumours and choroid plexus papillomas. Anaplastic ependymomas as well as choroid plexus carcinomas show decreased FOXJ1 expression and its associated ciliogenesis programme genes. In ependymomas and choroid plexus tumours, reduced expression of FOXJ1 and its ciliogenesis programme are markers of poor outcome and are therefore useful biomarkers for assessing these tumours. Transitions in ciliogenesis define distinct differentiation states in ependymal and choroid plexus tumours with important implications for patient care. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Forkhead box J1 expression is upregulated and correlated with prognosis in patients with clear cell renal cell carcinoma.

The forkhead box (FOX) family of transcription factors are considered to have a role in tumorigenesis. FOXJ1 is a member of the FOX family; however, its function in human renal cell carcinoma (RCC) has remained to be elucidated. Therefore, the present study evaluated the expression of FOXJ1 in human clear cell RCC and the effect of FOXJ1 on the proliferative ability of RCC cells. The RCC specimens analyzed in the present study were obtained from 286 patients with RCC who underwent nephrectomy. FOXJ1 mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction, and FOXJ1 protein expression levels were determined using immunohistochemistry and western blot analysis. To determine the effect of FOXJ1 on the proliferative ability of RCC cells, the expression of FOXJ1 was decreased using small interfering (si)RNA, and a FOXJ1 vector was stably transfected into RCC cell lines. The proliferative ability of RCC cells was then examined using a WST-1 assay and xenograft experiments with BALB/c nude mice, where the association between FOXJ1 expression and patient survival was determined using Kaplan-Meier analysis. FOXJ1 expression was significantly higher in RCC tissues compared with that of healthy renal tissues. Furthermore, FOXJ1 expression was associated with tumor stage, histologic grade and size. In addition, FOXJ1 significantly enhanced the proliferation of RCC cells in vitro and in vivo. The present study identified that FOXJ1 expression was upregulated in RCC and enhanced the proliferative ability of RCC cells. Therefore, FOXJ1 may serve as an independent prognostic marker and a therapeutic target for the treatment of patients with RCC.

ATP4 and ciliation in the neuroectoderm and endoderm of Xenopus embryos and tadpoles.

During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/β-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516–527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/β-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/β-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.

ATP4a is required for development and function of the Xenopus mucociliary epidermis – a potential model to study proton pump inhibitor-associated pneumonia

Proton pump inhibitors (PPIs), which target gastric H+/K+ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/β-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways.

Prostaglandin signaling in ciliogenesis during development

Prostaglandin signaling in ciliogenesis during development

PL-02 * NOTCH-MEDIATED SUPPRESSION OF MULTICILIATE DIFFERENTIATION PROMOTES CHOROID PLEXUS TUMOR INITIATION FROM EPITHELIAL PROGENITOR IN RESPONSE TO Shh SIGNALS

Choroid plexus (CP) tumors are intraventricular papillary neoplasms mainly found in children. While CP papilloma is a benign tumor, CP carcinoma is malignant and associated with poor prognosis. Though previous studies implicate Notch signaling in CP tumorigenesis, the developmental origin and mechanisms of Notch-induced CP tumors remain unclear. We used the Rosa26-NICD1 strain to express the intracellular domain of Notch 1 (NICD1) using Cre-loxP technologies. Hindbrain CP targeted for NICD1 expression developed abnormal growth consistent with CP papilloma. While Notch-induced CP tumor cells expressed CP markers Lmx1a and Otx2, the expression of Aquaporin 1 (AQP1), marker for differentiated CP epithelium, was absent. Embryonic studies showed that CP tumor arises during development and exhibit properties similar to those of CP epithelial progenitors. However, unlike progenitor cells, tumor cells fail to exit the cell cycle and differentiate into mature CP epithelium. Gene expression studies revealed increased expression of Gli1 and N-Myc, effectors of the Sonic Hedgehog (Shh) pathway, in both Notch-induced CP tumors and human CP tumors, suggesting the role of Shh signaling in CP tumors. Conversely, Shh treatment stimulated the proliferation of Notch-induced CP tumor cells. Vertebrate Shh signaling is uniquely dependent on primary cilium. Unlike differentiated CP epithelial cells with small tufts of primary cilia on their apical surface, Notch-induced CP tumor cells only possess solitary primary cilium. Further analysis revealed reduced expression of Foxj1 and Mcidas, crucial regulators of multiciliate differentiation, in tumor cells. Together, these results indicate that Notch-induced CP tumor arises from CP epithelial progenitors during development but lack terminal differentiation; and Notch pathway activation in tumor cells suppresses multiciliate differentiation, thereby allowing Shh-driven proliferation. Our studies demonstrate that Notch and Shh pathways converge on primary cilium to drive CP tumor formation, Shh pathway is a potential therapeutic target for Notch-induced CP tumors.

Abstract 3095: Notch-induced choroid plexus tumor arises from epithelial progenitor and depends on sonic hedgehog signaling for growth

Abstract Introduction: Choroid plexus tumors are rare intraventricular papillary neoplasms arising from choroid plexus (CP) epithelium. They occur most often in childhood and comprise 10-20% of all brain tumors in infants. Choroid plexus papilloma is a benign tumor, whereas choroid plexus carcinoma is malignant and most commonly found in pediatric population. Knowledge of the oncogenic processes in CP tumor formation will profoundly impact the development of new and molecular targeted therapies. Previous studies implicate Notch signaling in CP tumorigenesis. However, mechanisms underlying Notch-driven CP tumor formation remain unclear. Animal models of CP tumors have great potential to facilitate the discovery and development of molecular diagnostics and targeted therapies for these rare diseases. Methods and Results: We used the Rosa26-NICD1 strain which will constitutively express the intracellular domain of Notch 1 (NICD1) from the Rosa26 locus after Cre-mediated DNA recombination. The hindbrain CP targeted for NICD1 expression was significantly enlarged with well circumscribed, globular, cauliflower like masses. Histological analysis revealed that CP from NICD1-expressing mutant mice contained an abnormal growth in the 4th ventricle consistent with properties of choroid plexus tumor. Tumor cells exhibit enhanced proliferation transiently after birth: tumor cell proliferation was highest at birth, then gradually decreased and completely stopped at 2 weeks after birth. While CP tumor cells expressed early CP lineage markers such as Lmx1a and Otx2, they didn’t express Aquaporin 1 (AQP1), marker for differentiated CP epithelial cells, suggesting lack of terminal differentiation. We further showed that CP tumor arises during development as ectopic Lmx1a+ cells in CP and exhibit properties similar to those of CP epithelium progenitors. However, unlike progenitor cells, tumor cells fail to exit the cell cycle and differentiate into mature epithelial cells. We characterized changes in gene expression in tumor cells by quantitative RT-PCR. Surprisingly, we detected increased levels of Shh, Gli1 and N-Myc, effectors of the Sonic Hedgehog (Shh) pathway, in proliferating tumor cells. CP tumor cells cultured in serum-free conditions underwent rapid proliferation in the presence of Shh. Ultra-structural studies revealed lack of multi-ciliation differentiation in tumors cells, whereas the expression of Foxj1 and Midas, crucial regulators of cilia differentiation, was significantly downregulated. Conclusions: We developed a genetic model of CP tumor driven by Notch signaling activation. Notch 1-induced CP tumor arises during development from CP epithelial progenitors but lack terminal differentiation and multi-ciliation of mature CP epithelial cells. The proliferation of Notch 1-induced tumors is supported by Shh pathway, indicating Shh pathway as a therapeutic target for Notch-driven CP tumors. Citation Format: Haotian Zhao, Li Li, Katie Picotte. Notch-induced choroid plexus tumor arises from epithelial progenitor and depends on sonic hedgehog signaling for growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2014-3095

Decreased expression of FOXJ1 is a potential prognostic predictor for progression and poor survival of gastric cancer.

FOXJ1 is a member of the forkhead transcription factor family, which has been mostly studied for its role in the development of ciliated epithelium and immunology. However, the role of FOXJ1 in tumorigenesis remains largely unknown or even conflicting. We thus investigated FOXJ1 expression in gastric cancer and analyzed its correlations with tumor progression and prognosis. The expression of FOXJ1 was detected by immunohistochemistry in 105 gastric cancer samples and adjacent noncancerous tissues. Staining evaluation was conducted to assess clinicopathological parameters and the survival rate. In addition, the relation between FOXJ1 and metastasis was investigated in another 40 pairs of primary lesions and corresponding lymph node metastases. Furthermore, cell proliferation, migration, and invasion were confirmed in vitro. Decreased FOXJ1 expression was significantly correlated with clinic stage, lymph node metastasis, and distant metastasis, and lower FOXJ1 expression independently predicted shorter survival time in gastric carcinoma. Moreover, the positive incidence of FOXJ1 decreased significantly in metastatic lymph nodes compared with that in the primary lesions. Consistently, FOXJ1 overexpression significantly weakened cell proliferation, motility, migration, and invasion, while FOXJ1 knockdown induced the opposite effects. Decreased expression of FOXJ1 is an independent prognostic predictor for gastric cancer and is critical to disease progression. FOXJ1 may be an attractive therapeutic target for the treatment of gastric cancer.

Generation of multiciliated cells in functional airway epithelium from human iPSC (1094.5)

Despite a significant improvement in the availability of therapeutic options to treat lung diseases, pulmonary disease still remains a major cause of morbidity and mortality around the world. Currently there are limited opportunities to study human lung disease either in vivo and in vitro. Using induced pluripotent stem cells (iPSC) we have generated a reproducible differentiation protocol to make mature post‐mitotic multiciliated cells in a functional airway epithelium.iPSC were generated from human skin biopsies and differentiated via FOXA2+SOX17+ definitive endoderm (>90% efficiency) to FOXA2+NKx2.1+ anterior foregut endoderm, FOXA2+NKx2.1+SOX2+ (~50% efficiency) pulmonary endoderm and then matured in an air liquid interface. Robust multiciliogenesis occurred when Notch signaling was inhibited and was confirmed by; i) the assembly of multiple pericentrin stained centrioles at the apical surface, ii) expression of transcription factor FOXJ1 and iii) presence of multiple acetylated tubulin labeled cilia projections in individual cells. The presence of NKx2.1+CC10+ Clara cells, MUC5A/C+ goblet cells and FOXA2+p63+ basal cells was also confirmed showing we are generating a complete polarized epithelial cell layer comprised of all relevant cell types. Functional cAMP activated and CFTRinh‐172 sensitive CFTR currents were recorded in isolated epithelial cells by whole cell patch clamp technique. Furthermore, we have corrected the deltaF508 mutation in the CFTR gene (>80% of all cases of CF) using a combination of CRISPR‐Cas9 endonuclease‐mediated genome editing and piggyBac transposase technologies, in the CF patient‐derived iPSC.The generation of mature multiciliated cells in a human iPSC differentiated respiratory epithelium and the ability to correct disease causing mutations provides a significant advancement toward modeling a number of human respiratory diseases in vitro.Grant Funding Source: Supported in part by CIRM and the Berger Foundation

RFX7 is required for the formation of cilia in the neural tube

Regulatory Factor X (RFX) transcription factors are important for development and are likely involved in the pathogenesis of serious human diseases including ciliopathies. While seven RFX genes have been identified in vertebrates and several RFX transcription factors have been reported to be regulators of ciliogenesis, the role of RFX7 in development including ciliogenesis is not known. Here we show that RFX7 in Xenopus laevis is expressed in the neural tube, eye, otic vesicles, and somites. Knockdown of RFX7 in Xenopus embryos resulted in a defect of ciliogenesis in the neural tube and failure of neural tube closure. RFX7 controlled the formation of cilia by regulating the expression of RFX4 gene, which has been reported to be required for ciliogenesis in the neural tube. Moreover, ectopic expression of Foxj1, which is a master regulator of motile cilia formation, suppressed the expression of RFX4 but not RFX7. Taken together, RFX7 plays an important role in the process of neural tube closure at the top of the molecular cascade which controls ciliogenesis in the neural tube.