Transcription profiling of mouse model of islet dysmorphogenesis
ABSTRACT: In the past decade, several transcription factors critical for pancreas development have been identified. Despite this success, many of the cell surface and extracellular factors necessary for proper islet morphogenesis and function remain uncharacterized. Previous studies have shown that transgenic over-expression of the transcription factor HNF6 specifically in the pancreatic endocrine cell lineage resulted in the disruption of islet morphogenesis, including dysfunctional endocrine cell sorting, increased islet size, and failure of islets to migrate away from the ductal epithelium. We exploited the dysmorphic islets in pdx1PBHnf6 animals as a tool to identify factors important for islet morphogenesis. Genome-wide microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal pancreas tissue from wild type and pdx1PBHnf6 animals. We report the identification of genes with an altered expression in HNF6 Tg animals and highlight factors with potential importance in islet morphogenesis. Experiment Overall Design: Whole genome microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal pancreas tissue from wild type (WT) animals and HNF6 transgenic animals at a time when the events of normal islet morphogenesis are occurring (e18.5 and postnatal day 1). RNA was isolated from individual pancreata at the appropriate developmental stage. Highly pure samples were pooled according to their genotype (3-5 animals per pool) in order to obtain an adequate quantity of RNA for Affymetrix GeneChip analysis. Twelve samples were run in total, 3 replicates each of 4 groups: embryonic day 18.5, wild type mouse; embryonic day 18.5, HNF6 Transgenic mouse; post-natal day 1, wild type mouse; post-natal day 1, HNF6 Transgenic mouse.
Project description:RNA-seq analysis of RNA from embryonic day 18.5 pancreas Overall design: pancreas mRNA profiles of E18.5 C57Bl/6 mice were generated by deep sequencing using an Illumina Hiseq 2500.
Project description:Purpose: During postnatal development the fetal skeletal muscle undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS) Methods:We performed RNA-seq for high-resolution analysis of transcriptome changes during postnatal mouse skeletal muscle development using RNA from gastrocnemius muscle from embryonic day 18.5, postnatal day 2 (PN2), PN14, PN28, adult (22 weeks). Results: We identified a novel role for AS in several cellular functions including calcium handling and membrane organization during postnatal skeletal muscle development. Furthermore, AS transitions within calcium handling genes are responsive to Celf1 and Mbnl1, RNA-binding proteins with developmentally regulated expression. Conclusions: We identified a novel role for AS in several functional categories during postnatal development including calcium handling, vesicular trafficking and cell junctions. Overall design: Mouse skeletal muscle mRNA profiles of embryonic day 18.5, postnatal day 2 (PN2), PN14, PN28, adult (22 weeks) gastrocnemius muscle were generated by deep sequencing using Illumina HiSeq 2500.
Project description:In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, likely mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell elongation. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons in vertebrates. Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling, and conclude that Islet is a key regulatory factor governing morphogenesis of the palps. Three biological replicates were analyzed for both sample types (Fox positive and Negative).
Project description:Molecular characterization of PERK's role in the mouse exocrine pancreas. Postnatal days 16, 19 and 32 were examined. Postnatal day 16 represents a stage immediately prior to severe cytological changes in the exPKO mice. Postnatal day 19 represents the initial stage of severe phenotypes in the exPKO mice. Postnatal day 32 represents the middle stage of severe phenotypes (about 2 wks from the initiation) in the exPKO mice. Experiment Overall Design: Comparison between the two genotypes (Perk mutant vs. wild-type) at postnatal days 16, 19 and 32.
Project description:Early postnatal overnutrition causes persistent dysregulation of endocrine pancreas function. We used genome-scale DNA methylation profiling in the suckling-period small litter (SL) mouse model to test whether this occurs via persistent epigenetic changes in pancreatic islets. Although SL islets showed DNA methylation changes directly after weaning and in adulthood, few of these were present at both ages, contrary to our hypothesis. Most interestingly, we discovered that genomic regions that are hypermethylated in exocrine relative to endocrine pancreas tend to gain methylation in islets during aging. Focusing on a subset of genes relevant to β cell function, we showed that these methylation differences are strongly correlated with expression. Together, our results provide the novel insight that DNA methylation changes that occur as islets age indicate an overall epigenetic drift toward the exocrine pancreas epigenome. These concerted shifts in the islet methylome could contribute to the age-associated decline in endocrine pancreas function. Pancreatic islets were isolated from P21/P180 SL or C mice. To ensure purity of islets, 3 rounds of manual picking were performed in each samples. Whole pancreas samples, ~98% of which is exocrine pancreas, were used as exocrine pancreas. There are 5 mice per group.
Project description:We address the function of HNF6 in the mouse liver metabolism and Rev-erba cistrome Overall design: We performed Rev-erba ChIP-seq in mouse livers at 5pm of the day and compared between WT and HNF6-depleted livers.
Project description:This study determines pineal gland gene expression levels in the NeuroD1 knockout mouse at postnatal day zero. Comparison was performed against pineal gland gene expression levels in 129 wildtype mice also disected at P0. Keywords: Comparison of wildtype versus transgenic pineal gland gene expression Overall design: Wildype 129 mice served as the reference in comparing the levels of gene expression in the NeuroD1 transgenic animals. 3 pineal glands were disected at P0 during the daytime and pooled for each sample. 3 separate biological samplings were performed. Triplicate arrays were run for wildtype and the homozygous animals.
Project description:Comparison of two microarray platforms: the Mouse PancChip 5.0 and the Affymetrix GeneChip Mouse Genome 430 2.0 Array. The aim of this experiment was to determine the ability to identify differentially expressed genes in islet and pancreas RNA, and the sensitivity of the two platforms, using the same source material in a carefully controlled manner. RNA was extracted from adult mouse pancreas (n=5) and highly purified islet samples (n = 5). All samples were amplified once. 5 biological replicates (islets vs. pancreas) in a dye swap experimental design were hybridized to the PancChip. 3 biological replicates, using the same amplified RNA hybridized to the PancChip, of both the pancreas and islet samples were also hybridized to the GeneChip. All data were normalized using appropriate methods and differential expression between islet and pancreas was determined using PaGE with a 10% FDR. The study revealed that the PancChip is a highly cost effective alternative to the Affymetrix 430-2, that the PancChip is up to 60% more sensitive than the Affymetrix 430-2 GeneChip and that 80% (7,000) of the probe sets on the PancChip show expression in either Islets or Pancreas, while only 25% (6,800) show expression on the Affymetrix GeneChip.
Project description:We address the function of HNF6 in the mouse liver metabolism and Rev-erba cistrome We performed microarray in mouse livers at 5pm of the day and compared between WT and HNF6-depleted livers.