Deep learning approach identified a gene signature predictive of the severity of renal damage caused by chronic cadmium accumulation.

Epidemiology studies have indicated that environmental cadmium exposure, even at low levels, will result in chronic cadmium accumulation in the kidney with profound adverse consequences and that the diabetic population is more susceptible. However, the underlying mechanisms are yet not fully understood. In the present study, we applied an animal model to study chronic cadmium exposure-induced renal injury and performed whole transcriptome profiling studies. Repetitive CdCl2 exposure resulted in cadmium accumulation and remarkable renal injuries in the animals. The diabetic ob/ob mice manifested increased severity of renal injury compared with the wild type C57BL/6 J littermate controls. RNA-Seq data showed that cadmium treatment induced dramatic gene expression changes in a dose-dependent manner. Among the differentially expressed genes include the apoptosis hallmark genes which significantly demarcated the treatment effects. Pathway enrichment and network analyses revealed biological oxidation (mainly glucuronidation) as one of the major stress responses induced by cadmium treatment. We next implemented a deep learning algorithm in conjunction with cloud computing and discovered a gene signature that can predict the degree of renal injury induced by cadmium treatment. The present study provided, for the first time, a comprehensive mechanistic understanding of chronic cadmium-induced nephrotoxicity in normal and diabetic populations at the whole genome level.

Chemical genetic-based phenotypic screen reveals novel regulators of gluconeogenesis in human primary hepatocytes.

Insulin resistance is a pathophysiological hallmark of type 2 diabetes and nonalcoholic fatty liver disease. Under the condition of fat accumulation in the liver, suppression of hepatic glucose production by insulin is diminished. In order to gain deeper understanding of dysregulation of glucose production in metabolic diseases, in the present study, we performed an unbiased phenotypic screening in primary human hepatocytes to discover novel mechanisms that regulate gluconeogenesis in the presence of insulin. To optimize phenotypic screening process, we used a chemical genetic screening approach by building a small-molecule library with prior knowledge of activity-based protein profiling. The "positive hits" result from the screen will be small molecules with known protein targets. This makes downstream deconvolution process (i.e., determining the relevant biological targets) less time-consuming. To unbiasedly decipher the molecular targets, we developed a novel statistical method and discovered a set of genes, including DDR3 and CACNA1E that suppressed gluconeogenesis in human hepatocytes. Further investigation, including transcriptional profiling and gene network analysis, was performed to understand the molecular functions of DRD3 and CACNA1E in human hepatocytes.

A circulating microRNA signature as noninvasive diagnostic and prognostic biomarkers for nonalcoholic steatohepatitis.

BACKGROUND: Noninvasive biomarkers are urgently needed for patients with nonalcoholic steatohepatitis (NASH) to assist in diagnosis, monitoring disease progression and assessing treatment response. Recently several exploratory studies showed that circulating level of microRNA is associated with NASH and correlated with disease severity. Although these data were encouraging, the application of circulating microRNA as biomarkers for patient screening and stratification need to be further assessed under well-controlled conditions. RESULTS: The expression of circulating microRNAs were profiled in diet-induced NASH progression and regression models to assess the diagnostic and prognostic values and the translatability between preclinical mouse model and men. Since these mice had same genetic background and were housed in the same conditions, there were minimal confounding factors. Histopathological lesions were analyzed at distinct disease progression stages along with microRNA measurement which allows longitudinal assessment of microRNA as NASH biomarkers. Next, differentially expressed microRNAs were identified and validated in an independent cohorts of animals. Thirdly, these microRNAs were examined in a NASH regression model to assess whether they would respond to NASH treatment. MicroRNA profiling in two independent cohorts of animals validated the up-regulation of 6 microRNAs (miR-122, miR-192, miR-21, miR-29a, miR-34a and miR-505) in NASH mice, which was designated as the circulating microRNA signature for NASH. The microRNA signature could accurately distinguish NASH mice from lean mice, and it responded to chow diet treatment in a NASH regression model. To further improve the performance of microRNA-based biomarker, a new composite biomarker was proposed, which consists of miR-192, miR-21, miR-505 and ALT. The new composite biomarker outperformed the microRNA signature in predicting NASH mice which had NAS > 3, and deserves further validations in large scale studies. CONCLUSION: The present study supported the translation of circulating microRNAs between preclinical models and humans in NASH pathogenesis and progression. The microRNA-based composite biomarker may be used for non-invasive diagnosis, clinical monitoring and assessing treatment response for NASH.

Histone methyltransferase SETDB1 regulates liver cancer cell growth through methylation of p53.

SETDB1 is a histone H3K9 methyltransferase that has a critical role in early development. It is located within a melanoma susceptibility locus and facilitates melanoma formation. However, the mechanism by which SETDB1 regulates tumorigenesis remains unknown. Here we report the molecular interplay between SETDB1 and the well-known hotspot gain-of-function (GOF) TP53 R249S mutation. We show that in hepatocellular carcinoma (HCC) SETDB1 is overexpressed with moderate copy number gain, and GOF TP53 mutations including R249S associate with this overexpression. Inactivation of SETDB1 in HCC cell lines bearing the R249S mutation suppresses cell growth. The TP53 mutation status renders cancer cells dependent on SETDB1. Moreover, SETDB1 forms a complex with p53 and catalyses p53K370 di-methylation. SETDB1 attenuation reduces the p53K370me2 level, which subsequently leads to increased recognition and degradation of p53 by MDM2. Together, we provide both genetic and biochemical evidence for a mechanism by which SETDB1 regulates cancer cell growth via methylation of p53.

SETDB1 modulates PRC2 activity at developmental genes independently of H3K9 trimethylation in mouse ES cells.

SETDB1, a histone methyltransferase responsible for methylation of histone H3 lysine 9 (H3K9), is involved in maintenance of embryonic stem (ES) cells and early embryonic development of the mouse. However, how SETDB1 regulates gene expression during development is largely unknown. Here, we characterized genome-wide SETDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SETDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SETDB1 solo peaks, particularly those near neural development-related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins JARID2 and MTF2. Genetic deletion of Setdb1 reduced EZH2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SETDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SETDB1 methyltransferase activity. The currently identified reciprocal action between SETDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation.

G9a is transactivated by C/EBPß to facilitate mitotic clonal expansion during 3T3-L1 preadipocyte differentiation.

In 3T3-L1 preadipocyte differentiation, the CCAAT/enhancer-binding protein-ß (C/EBPß) is an important early transcription factor that activates cell cycle genes during mitotic clonal expansion (MCE), sequentially activating peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα during terminal differentiation. Although C/EBPß acquires its DNA binding activity via dual phosphorylation at about 12-16 h postinduction, the expression of PPARγ and C/EBPα is not induced until 36-72 h. The delayed expression of PPARγ and C/EBPα ensures the progression of MCE, but the mechanism responsible for the delay remains elusive. We provide evidence that G9a, a major euchromatic methyltransferase, is transactivated by C/EBPß and represses PPARγ and C/EBPα through H3K9 dimethylation of their promoters during MCE. Inhibitor- or siRNA-mediated G9a downregulation modestly enhances PPARγ and C/EBPα expression and adipogenesis in 3T3-L1 preadipocytes. Conversely, forced expression of G9a impairs the accumulation of triglycerides. Thus, this study elucidates an epigenetic mechanism for the delayed expression of PPARγ and C/EBPα.

Involvement of histone acetylation of Sox17 and Foxa2 promoters during mouse definitive endoderm differentiation revealed by microRNA profiling.

Generation of hepatocyte from embryonic stem cells (ESCs) holds great promise for hepatocyte replacement therapy to treat liver diseases. Achieving high efficiency of directed differentiation of ESCs to hepatocyte is of critical importance. Previously, Wnt3a has been reported to promote Activin A-induced human definitive endoderm (DE) differentiation, the early stage of hepatocyte differentiation. However, the underlying molecular mechanisms are not clear. Growing evidence demonstrated that microRNAs (miRNAs) are key regulators involved in various important biological processes including the regulation of stem cell differentiation. In the present study, we profiled genome wide miRNA expression during Wnt3a and Activin A induced mouse DE differentiation. We uncovered distinct miRNA expression patterns during DE differentiation with the identification of a subset of miRNAs whose expression is synergistically regulated by Wnt3a/Activin A treatment at different stages of DE differentiation. Forced expression of a pool of such synergistically regulated miRNAs alone could partially promote DE differentiation, indicating a regulatory role of them. Using TargetScan and GeneGO pathway analyses, the synergistically regulated miRNAs are predicted to regulate key pathways involved in DE differentiation; among them includes the regulation of histone acetylation. Consistently, Wnt3a and Activin A treatment increased global histone acetylation which can be partially mimicked by over expression of the pooled miRNAs. Chromatin IP (ChIP) experiments demonstrated that the promoter regions of Sox17 and Foxa2 are subjected to histone acetylation regulation. Administration of Hdac inhibitors greatly augmented DE differentiation. Our data uncovered a novel epigenetic mechanism of Wnt3a and Activin A induced DE differentiation, whereby the treatment of growth factors induced histone acetylation at least in part by the regulation of miRNA expression.

Developing and applying a gene functional association network for anti-angiogenic kinase inhibitor activity assessment in an angiogenesis co-culture model.

BACKGROUND: Tumor angiogenesis is a highly regulated process involving intercellular communication as well as the interactions of multiple downstream signal transduction pathways. Disrupting one or even a few angiogenesis pathways is often insufficient to achieve sustained therapeutic benefits due to the complexity of angiogenesis. Targeting multiple angiogenic pathways has been increasingly recognized as a viable strategy. However, translation of the polypharmacology of a given compound to its antiangiogenic efficacy remains a major technical challenge. Developing a global functional association network among angiogenesis-related genes is much needed to facilitate holistic understanding of angiogenesis and to aid the development of more effective anti-angiogenesis therapeutics. RESULTS: We constructed a comprehensive gene functional association network or interactome by transcript profiling an in vitro angiogenesis model, in which human umbilical vein endothelial cells (HUVECs) formed capillary structures when co-cultured with normal human dermal fibroblasts (NHDFs). HUVEC competence and NHDF supportiveness of cord formation were found to be highly cell-passage dependent. An enrichment test of Biological Processes (BP) of differentially expressed genes (DEG) revealed that angiogenesis related BP categories significantly changed with cell passages. Built upon 2012 DEGs identified from two microarray studies, the resulting interactome captured 17226 functional gene associations and displayed characteristics of a scale-free network. The interactome includes the involvement of oncogenes and tumor suppressor genes in angiogenesis. We developed a network walking algorithm to extract connectivity information from the interactome and applied it to simulate the level of network perturbation by three multi-targeted anti-angiogenic kinase inhibitors. Simulated network perturbation correlated with observed anti-angiogenesis activity in a cord formation bioassay. CONCLUSION: We established a comprehensive gene functional association network to model in vitro angiogenesis regulation. The present study provided a proof-of-concept pilot of applying network perturbation analysis to drug phenotypic activity assessment.

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity.

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge.

Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.

Analysis of pathway activity in primary tumors and NCI60 cell lines using gene expression profiling data.

To determine cancer pathway activities in nine types of primary tumors and NCI60 cell lines, we applied an in silica approach by examining gene signatures reflective of consequent pathway activation using gene expression data. Supervised learning approaches predicted that the Ras pathway is active in approximately 70% of lung adenocarcinomas but inactive in most squamous cell carcinomas, pulmonary carcinoids, and small cell lung carcinomas. In contrast, the TGF-beta, TNF-alpha, Src, Myc, E2F3, and beta-catenin pathways are inactive in lung adenocarcinomas. We predicted an active Ras, Myc, Src, and/or E2F3 pathway in significant percentages of breast cancer, colorectal carcinoma, and gliomas. Our results also suggest that Ras may be the most prevailing oncogenic pathway. Additionally, many NCI60 cell lines exhibited a gene signature indicative of an active Ras, Myc, and/or Src, but not E2F3, beta-catenin, TNF-alpha, or TGF-beta pathway. To our knowledge, this is the first comprehensive survey of cancer pathway activities in nine major tumor types and the most widely used NCI60 cell lines. The "gene expression pathway signatures" we have defined could facilitate the understanding of molecular mechanisms in cancer development and provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screening.

Changes in osteoblast, chondrocyte, and adipocyte lineages mediate the bone anabolic actions of PTH and small molecule GSK-3 inhibitor.

Parathyroid hormone (PTH) and glycogen synthase kinase-3 (GSK-3) inhibitor 603281-31-8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281-31-8 in rat osteopenia model. Female 6-month-old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1-38) at 10 microg/kg/day s.c. or 603281-31-8 at 3 mg/kg/day p.o. for 60 days. Twenty-four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P<0.05) were subjected to pathway analysis to delineate relevant bio-processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK-3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three-fold increase in marrow adiposity. PTH and GSK-3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK-3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU-fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK-3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes.

DNA microarray data integration by ortholog gene analysis reveals potential molecular mechanisms of estrogen-dependent growth of human uterine fibroids.

BACKGROUND: Uterine fibroids or leiomyoma are a common benign smooth muscle tumor. The tumor growth is well known to be estrogen-dependent. However, the molecular mechanisms of its estrogen-dependency is not well understood. METHODS: Differentially expressed genes in human uterine fibroids were either retrieved from published papers or from our own statistical analysis of downloaded array data. Probes for the same genes on different Affymetrix chips were mapped based on probe comparison information provided by Affymetrix. Genes identified by two or three array studies were submitted for ortholog analysis. Human and rat ortholog genes were identified by using ortholog gene databases, HomoloGene and TOGA and were confirmed by synteny analysis with MultiContigView tool in the Ensembl genome browser. RESULTS: By integrated analysis of three recently published DNA microarray studies with human tissue, thirty-eight genes were found to be differentially expressed in the same direction in fibroid compared to adjacent uterine myometrium by at least two research groups. Among these genes, twelve with rat orthologs were identified as estrogen-regulated from our array study investigating uterine expression in ovariectomized rats treated with estrogen. Functional and pathway analyses of the twelve genes suggested multiple molecular mechanisms for estrogen-dependent cell survival and tumor growth. Firstly, estrogen increased expression of the anti-apoptotic PCP4 gene and suppressed the expression of growth inhibitory receptors PTGER3 and TGFBR2. Secondly, estrogen may antagonize PPARgamma signaling, thought to inhibit fibroid growth and survival, at two points in the PPAR pathway: 1) through increased ANXA1 gene expression which can inhibit phospholipase A2 activity and in turn decrease arachidonic acid synthesis, and 2) by decreasing L-PGDS expression which would reduce synthesis of PGJ2, an endogenous ligand for PPARgamma. Lastly, estrogen affects retinoic acid (RA) synthesis and mobilization by regulating expression of CRABP2 and ALDH1A1. RA has been shown to play a significant role in the development of uterine fibroids in an animal model. CONCLUSION: Integrated analysis of multiple array datasets revealed twelve human and rat ortholog genes that were differentially expressed in human uterine fibroids and transcriptionally responsive to estrogen in the rat uterus. Functional and pathway analysis of these genes suggest multiple potential molecular mechanisms for the poorly understood estrogen-dependent growth of uterine fibroids. Fully understanding the exact molecular interactions among these gene products requires further study to validate their roles in uterine fibroids. This work provides new avenues of study which could influence the future direction of therapeutic intervention for the disease.

Comparative analysis and integrative classification of NCI60 cell lines and primary tumors using gene expression profiling data.

BACKGROUND: NCI60 cell lines are derived from cancers of 9 tissue origins and have been invaluable in vitro models for cancer research and anti-cancer drug screen. Although extensive studies have been carried out to assess the molecular features of NCI60 cell lines related to cancer and their sensitivities to more than 100,000 chemical compounds, it remains unclear if and how well these cell lines represent or model their tumor tissues of origin. Identification and confirmation of correct origins of NCI60 cell lines are critical to their usage as model systems and to translate in vitro studies into clinical potentials. Here we report a direct comparison between NCI60 cell lines and primary tumors by analyzing global gene expression profiles. RESULTS: Comparative analysis suggested that 51 of 59 cell lines we analyzed represent their presumed tumors of origin. Taking advantage of available clinical information of primary tumor samples used to generate gene expression profiling data, we further classified those cell lines with the correct origins into different subtypes of cancer or different stages in cancer development. For example, 6 of 7 non-small cell lung cancer cell lines were classified as lung adenocarcinomas and all of them were classified into late stages in tumor progression. CONCLUSION: Taken together, we developed and applied a novel approach for systematic comparative analysis and integrative classification of NCI60 cell lines and primary tumors. Our results could provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screenings. Moreover, this gene expression profile based approach can be generally applied to evaluate experimental model systems such as cell lines and animal models for human diseases.

Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model.

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.

Optimization and validation of small quantity RNA profiling for identifying TNF responses in cultured human vascular endothelial cells.

INTRODUCTION: Affymetrix oligonucleotide microarrays are widely used in basic and applied research (Lander, E.S., (1999). Array of hope. Nature Genetics 21, 3-4; Lockhart, D.J. & Winzeler, E.A. (2000) Genomics, gene expression and DNA arrays. Nature 405, 827-836.) The need for a significant amount of starting RNA has limited its use in applications where the amount of RNA is limiting, such as with Laser Captured Microdissection (LCM), small biopsies, or peripheral blood in rodent models. To overcome this limitation, various RNA amplification and labeling methods have been described, however, further optimization and validation of these methods are needed. METHODS: Here we reported using the Arcturus technology to optimize amplification and labeling of small amounts of RNA for Affymetrix microarray studies. We assessed the technical feasibility and variation introduced by differences in starting RNA quantity and differences in technical performance by microarray hybridization. RESULTS: We demonstrated that the current approach is reliable to amplify as little as 40 ng total RNA, and it is suitable for Affymetrix studies yielding satisfactory quantitative chip performance. We also showed that differences in labeling methods contribute more to variation than the differences in starting RNA quantity per se. As a model, we studied the well-documented TNF-induced inflammatory responses in cultured human vascular endothelial cells. We were able to recapitulate the TNF-induced responses using small RNA sample profiling.

Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse.

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach to test the hypothesis that Fgf8 plays such a role in developing OE. In young embryos, Fgf8 RNA is expressed in the rim of the invaginating nasal pit (NP), in a small domain of cells that overlaps partially with that of putative OE neural stem cells later in gestation. In mutant mice in which the Fgf8 gene is inactivated in anterior neural structures, FGF-mediated signaling is strongly downregulated in both OE proper and underlying mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ (VNO), nasal cavity, forebrain, lower jaw, eyelids and pinnae. Analysis of mutants indicates that although initial NP formation is grossly normal, cells in the Fgf8-expressing domain undergo high levels of apoptosis, resulting in cessation of nasal cavity invagination and loss of virtually all OE neuronal cell types. These findings demonstrate that Fgf8 is crucial for proper development of the OE, nasal cavity and VNO, as well as maintenance of OE neurogenesis during prenatal development. The data suggest a model in which Fgf8 expression defines an anterior morphogenetic center, which is required not only for the sustenance and continued production of primary olfactory (OE and VNO) neural stem and progenitor cells, but also for proper morphogenesis of the entire nasal cavity.

Notch1-expressing cells are indispensable for prostatic branching morphogenesis during development and re-growth following castration and androgen replacement.

Notch expression is frequently associated with progenitor cells, and its function is crucial for development. Our recent work showing that Notch1 is selectively expressed in basal epithelial cells of the prostate and higher Notch1 expression during development suggests that Notch1-expressing cells may define progenitor cells in the prostate. To test this hypothesis, we have generated a transgenic mouse line in which the Notch1-expressing cells can be ablated in a controlled manner. Specific targeting was achieved by expressing the bacterial nitroreductase, an enzyme that catalyzes its substrate into a cytotoxin capable of inducing apoptosis, under the Notch1 promoter. Cell death in transgenic prostate was confirmed by histological analyses including terminal dUTP nick-end labeling and caspase 3 immunocytochemical staining. We evaluated the consequences of ablation of Notch1-expressing cells in two systems, organ culture of early postnatal prostates and re-growth of prostate in castrated mice triggered by hormone replacement. Our data show that elimination of Notch1-expressing cells inhibited the branching morphogenesis, growth, and differentiation of early postnatal prostate in culture and impaired prostate re-growth triggered by hormone replacement in castrated mice. Furthermore, we found that Notch1 expression following castration and hormone replacement was concomitant with known basal cell markers p63 and cytokeratin 14 and was high in the proliferative human prostate epithelial cells. Taken together, these data suggest that Notch1-expressing cells define the progenitor cells in the prostatic epithelial cell lineage, which are indispensable for prostatic development and re-growth.

Robust generation of new hair cells in the mature mammalian inner ear by adenoviral expression of Hath1.

Although hair cells regenerate spontaneously in birds and lower vertebrates following injury, there is yet no effective way to stimulate hair cell regeneration in mature mammalian inner ears. Here we report that a large number of hair cells are produced in the sensory epithelium of cultured adult rat utricular maculae, via adenovirus-mediated overexpression of Hath1, a human atonal homolog. The generation of new hair cells via Hath1 expression does not involve cell proliferation based on bromodeoxyuridine immunocytochemistry. Furthermore, using a similar approach, hair cells are regenerated following aminoglycoside injury in these cultures. These data show conclusively that mature mammalian inner ears have the competence to produce a large number of new hair cells. Local adenoviral gene therapy in the inner ear may be a potential approach to treatment of hearing and balance disorders.