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Identification of differentially expressed genes in mouse Pax6 heterozygous lenses.

ABSTRACT: Pax6 is a critical regulator of the developing lens, other ocular tissues, central nervous system, and pancreas. Downstream targets of Pax6 are largely unknown. The present study was designed to identify differentially expressed genes in Pax6 heterozygous and normal mouse lenses.RNAs from 8-week-old normal and Pax6 heterozygous mouse lenses were analyzed by both RT-PCR differential display and a candidate-gene approach. The expression levels of identified genes were confirmed by semiquantitative RT-PCR.Eight transcripts encoding phosphatase inhibitor Pip-1 protein, heat shock protein Hsp40, Purkinje cell protein Pcp4, an expressed sequence tag (EST; AA331381) originally reported in a brain-specific library, Pitx3, and CBP, were confirmed to be downregulated in the Pax6 heterozygous mouse lenses. alphaB- and betaA3/A1-crystallin transcripts exhibited decreased expression in Pax6 heterozygous lenses, whereas the expression levels of other crystallins were virtually unchanged.The present data identify eight genes with expression levels that are decreased in Pax6 heterozygous lenses and provide evidence that four functional categories of transcripts-namely, small hsps (alphaB-crystallin and Hsp40), crystallins (alphaB- and betaA3/A1-crystallin), transcription factors (Pitx-3 and CBP), and components of signal transduction cascades (Pip-1) are under direct or indirect transcriptional control by Pax6.

SUBMITTER: Chauhan BK

PROVIDER: S-EPMC2080866 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Identification of differentially expressed genes in mouse Pax6 heterozygous lenses.

Chauhan Bharesh K BK Zhang Weiyan W Cveklova Kveta K Kantorow Marc M Cvekl Ales A

Investigative ophthalmology & visual science 20020601 6

<h4>Purpose</h4>Pax6 is a critical regulator of the developing lens, other ocular tissues, central nervous system, and pancreas. Downstream targets of Pax6 are largely unknown. The present study was designed to identify differentially expressed genes in Pax6 heterozygous and normal mouse lenses.<h4>Methods</h4>RNAs from 8-week-old normal and Pax6 heterozygous mouse lenses were analyzed by both RT-PCR differential display and a candidate-gene approach. The expression levels of identified genes we ...[more]

PMID: 12036994

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Project description:Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3 kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cell suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip. Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan(+) (osteoblasts), and DMP1topaz(+) (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz(+) and Col2.3cyan(+) cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz(+)cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz(+) cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz(+) cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz(+) and Col2.3cyan(+) cells in our study have already been assigned to bone development and physiology, for most of them we still lack any substantial data. Therefore, isolation of osteocyte and osteoblast cell populations and their subsequent microarray analysis allowed us to identify a number or genes and pathways with potential roles in regulation of bone mass.

Project description:BackgroundCurrent diagnosis and treatment of urinary bladder cancer (BC) has shown great progress with the utilization of microarrays.PurposeOur goal was to identify common differentially expressed (DE) genes among clinically relevant subclasses of BC using microarrays.Methodology/principal findingsBC samples and controls, both experimental and publicly available datasets, were analyzed by whole genome microarrays. We grouped the samples according to their histology and defined the DE genes in each sample individually, as well as in each tumor group. A dual analysis strategy was followed. First, experimental samples were analyzed and conclusions were formulated; and second, experimental sets were combined with publicly available microarray datasets and were further analyzed in search of common DE genes. The experimental dataset identified 831 genes that were DE in all tumor samples, simultaneously. Moreover, 33 genes were up-regulated and 85 genes were down-regulated in all 10 BC samples compared to the 5 normal tissues, simultaneously. Hierarchical clustering partitioned tumor groups in accordance to their histology. K-means clustering of all genes and all samples, as well as clustering of tumor groups, presented 49 clusters. K-means clustering of common DE genes in all samples revealed 24 clusters. Genes manifested various differential patterns of expression, based on PCA. YY1 and NFκB were among the most common transcription factors that regulated the expression of the identified DE genes. Chromosome 1 contained 32 DE genes, followed by chromosomes 2 and 11, which contained 25 and 23 DE genes, respectively. Chromosome 21 had the least number of DE genes. GO analysis revealed the prevalence of transport and binding genes in the common down-regulated DE genes; the prevalence of RNA metabolism and processing genes in the up-regulated DE genes; as well as the prevalence of genes responsible for cell communication and signal transduction in the DE genes that were down-regulated in T1-Grade III tumors and up-regulated in T2/T3-Grade III tumors. Combination of samples from all microarray platforms revealed 17 common DE genes, (BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3, ACTC1, MFAP4, SPARCL1, TAGLN, TPM2, CDC20, LHCGR, TM9SF1 and HCCS) 4 of which participate in numerous pathways.Conclusions/significanceThe identification of the common DE genes among BC samples of different histology can provide further insight into the discovery of new putative markers.

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Identification of differentially expressed genes in mouse Pax6 heterozygous lenses.

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Identification of differentially expressed genes in mouse Pax6 heterozygous lenses.

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