Comment[ArrayExpressAccession] E-GEOD-31004 Public Release Date 2011-12-15 Investigation Title Effects of Nicotine on the Fetal Mouse Palate Development and Transcriptome Comment[Submitted Name] Effects of Nicotine on the Fetal Mouse Palate Development and Transcriptome Experiment Description Nonsyndromic cleft palate is a common birth defect (1:700) with a complex etiology involving both genetic and environmental risk factors. Nicotine, a major teratogen present in tobacco products, was shown to cause alterations and delays in the developing fetus. To demonstrate the effect of nicotine on craniofacial development, particularly palatogenesis, we delivered three different doses of nicotine (1.5, 3.0 and 4.5 mg/kg/day) into pregnant BALB/c mice throughout their entire pregnancy using subcutaneous osmotic mini-pump. We assessed the pups for morphological anomalies, as well as genome-wide mRNA (transcriptome) microarray analysis. Consistent administration of nicotine caused developmental retardation, still birth, low birth weight, and significant palatal size and shape abnormality in the pups. However, it did not cause obvious cleft palate. The microarray data analysis using IPA identified differential expression of genes involved in various biological pathways, particularly cancer, genetic diseases, and tissue development in response to consistent nicotine exposure. 6232 up-regulated and 6310 down-regulated genes were detected in nicotine-treated groups compared to the control. Moreover, 45% of the genes associated with cleft palate were found to be affected by nicotine. Alterations of a subset of differentially expressed genes were illustrated with hierarchal clustering and RT-PCR. We concluded that consistent nicotine exposure during pregnancy interferes with normal growth and development of the fetus including palatogenesis; however, this interference does not result in cleft palate, rather smaller palate size with persistent MES. To our knowledge, this is the first experiment revealing the impact of nicotine on the fetal palate transcriptome in mice. Total 8 samples were analyzed. Using an osmotic minipump, duplicate samples from palates of either sterile physiological saline or nicotine (1.5 mg/kg/day, 3.0 mg/kg/day, or 4.5 mg/kg/day)-treated newborn pups. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Nawshad Ozturk Sheldon Aydemir Out Fung Nawshad Person First Name Ali Ferhat Elizabeth Serkan Hasan Eric Ali Person Mid Initials H Person Email anawshad@unmc.edu Person Affiliation Unviersity of Nebraska Medical Center Person Phone 402-472-1378 Person Fax 402-472-2551 Person Address Oral Biology, Unviersity of Nebraska Medical Center, 40th and Holdrege, Lincoln, NE, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE31004-1 P-GSE31004-6 P-GSE31004-3 P-GSE31004-8 P-GSE31004-7 P-GSE31004-2 P-GSE31004-4 P-GSE31004-5 Protocol Description ID_REF =
VALUE = dChip normalization The GeneChip arrays are hybridized overnight in the Affymetrix hybridization oven at 42oC. The Affymetrix GeneChip Fluidics Station 450 performs the automated Affymetrix wash and stain protocols. A small incision of 0.5 cm was made in the skin posterior to the shoulder of pregnant mice and an osmotic mini-pump (Model 1004 - Alzet Corp, CA) was implanted subcutaneously. The incision was closed with a wound clip and covered with an antibiotic. The clip was removed after 14 days and dams were allowed to give birth naturally at/around 21 days Scanned array images were analyzed by dChip applying a smoothing spline normalization method prior to obtaining model-based gene expression indices, a.k.a. signal values. There were no outliers identified by dChip, so all samples were carried on for subsequent analysis. When comparing two groups of samples to identify enriched genes in a given group, we used the lower confidence bound (LCB) of the fold change (FC) between the two groups as the cut-off criteria. If 90% LCB of FC between the two groups was above 1.2, the corresponding gene was considered to be differentially expressed. The Affymetrix GeneChip Scanner 3000 7G is used to generate the resultant GeneChip array image Sterile physiological saline or nicotine (1.5 mg/kg/day, 3.0 mg/kg/day, or 4.5 mg/kg/day) Total RNA was isolated from the posterior one-third of palatal tissues from newborn pups (n=2) using the RNeasy spin column RNA purification kit (Qiagen) The UNMC Microarray Core uses a variety of commercially available assays for generating biotin-labeled samples for hybridization to Affymetrix GeneChip Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name TREATMENT Experimental Factor Type treatment Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE31004 Comment[GEOLastUpdateDate] 2011-12-19 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-12-15 Comment[ArrayExpressSubmissionDate] 2011-07-26 SDRF File E-GEOD-31004.sdrf.txt