Genomic analyses identify molecular subtypes of pancreatic cancer.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.

Whole genomes redefine the mutational landscape of pancreatic cancer.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing.

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.

Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-ß, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Somatic point mutation calling in low cellularity tumors.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

qpure: A tool to estimate tumor cellularity from genome-wide single-nucleotide polymorphism profiles.

Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.

Deep-transcriptome and ribonome sequencing redefines the molecular networks of pluripotency and the extracellular space in human embryonic stem cells.

Recent RNA-sequencing studies have shown remarkable complexity in the mammalian transcriptome. The ultimate impact of this complexity on the predicted proteomic output is less well defined. We have undertaken strand-specific RNA sequencing of multiple cellular RNA fractions (>20 Gb) to uncover the transcriptional complexity of human embryonic stem cells (hESCs). We have shown that human embryonic stem (ES) cells display a high degree of transcriptional diversity, with more than half of active genes generating RNAs that differ from conventional gene models. We found evidence that more than 1000 genes express long 5' and/or extended 3'UTRs, which was confirmed by "virtual Northern" analysis. Exhaustive sequencing of the membrane-polysome and cytosolic/untranslated fractions of hESCs was used to identify RNAs encoding peptides destined for secretion and the extracellular space and to demonstrate preferential selection of transcription complexity for translation in vitro. The impact of this newly defined complexity on known gene-centric network models such as the Plurinet and the cell surface signaling machinery in human ES cells revealed a significant expansion of known transcript isoforms at play, many predicting possible alternative functions based on sequence alterations within key functional domains.

Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways.

The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as 'anchor' genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.

Redirection of renal mesenchyme to stromal and chondrocytic fates in the presence of TGF-beta2.

Many members of the transforming growth factor-beta (TGF-beta) superfamily have been shown to be important regulators of metanephric development. In this study, we characterized the effect of TGF-beta2 on metanephric development. Rat and mouse metanephroi cultured in the presence of exogenous TGF-beta2 for up to 15 days were small, and contained rudimentary ureteric branches and few glomeruli. These metanephroi were mostly comprised of mesenchymal cells, with two cell populations (designated Type 1 and Type 2 cells) evident. Type 1 cells were only observed when TGF-beta2 was added from the commencement of culture, they resembled chondroblasts and were Alcian Blue and Col IIB positive. Type 2 cells were observed whenever TGF-beta2 was added to the media, formed a band at the periphery of the explants consisting of 5-10 layers of spindle-shaped cells, and were alpha-smooth muscle actin positive. Molecular and RNA in situ hybridization analysis of metanephroi cultured in the presence of TGF-beta2 for 6 days demonstrated that Type 1 and 2 cells were negative for Pax2, WT1, GDNF and FoxD1. Gene expression profiling demonstrated an upregulation of chondrocyte, myogenic and stromal genes, some of which were identified as markers of Type 1 and Type 2 cells. In addition, TGF-beta2 was capable of maintaining the survival of mouse isolated metanephric mesenchyme (iMM) in the absence of serum or inductive signals from the ureteric epithelium. TGF-beta2 also induced the differentiation of iMM into Type 1 and 2 cells. The presence of chondrocytes and muscle in these cultures is reminiscent of the cell types found in some Wilms' tumors. These studies demonstrate that TGF-beta2 is capable of differentiating metanephric mesenchyme away from a renal cell fate.

RNA-MATE: a recursive mapping strategy for high-throughput RNA-sequencing data.

UNLABELLED: Mapping of next-generation sequencing data derived from RNA samples (RNAseq) presents different genome mapping challenges than data derived from DNA. For example, tags that cross exon-junction boundaries will often not map to a reference genome, and the strand specificity of the data needs to be retained. Here we present RNA-MATE, a computational pipeline based on a recursive mapping strategy for placing strand specific RNAseq data onto a reference genome. Maximizing the mappable tags can provide significant savings in the cost of sequencing experiments. This pipeline provides an automatic and integrated way to align color-space sequencing data, collate this information and generate files for examining gene-expression data in a genomic context. AVAILABILITY: Executables, source code, and exon-junction libraries are available from http://grimmond.imb.uq.edu.au/RNA-MATE/

Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment.

While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.

Stem cell transcriptome profiling via massive-scale mRNA sequencing.

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

Characterisation and trophic functions of murine embryonic macrophages based upon the use of a Csf1r-EGFP transgene reporter.

All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.

Characterization of the breast cancer associated ATM 7271T>G (V2424G) mutation by gene expression profiling.

Mutations in ATM are responsible for the autosomal recessive disorder ataxia telangiectasia. Heterozygous mutations in ATM have been associated with an elevated risk of breast cancer. We previously reported one breast cancer family in which ATM 7271T>G (V2424G) segregated with disease, and apparently acted in a dominant negative manner. We now report the screening of 782 multiple-case breast cancer families that identified two additional index cases with ATM 7271T>G. Phylogenetic sequence analysis showed that V2424 is a highly conserved residue, and that the 2424G variant is likely to interfere with function. To elucidate the consequences of this mutation, we expression profiled wild-type, heterozygous, and homozygous lymphoblastoid cell lines (LCLs) from Scottish and Australian families using an oligonucleotide microarray. Cluster analysis revealed 77 genes that were differentially expressed in homozygous and heterozygous V2424G cells (compared to wild-type) and 11 genes differentially expressed in the homozygous cells. We also evaluated the profiles of LCLs after exposure to ionizing radiation (IR) and identified 77 genes that were differentially expressed in wild-type cells, but not in homozygous or heterozygous V2424G cells. We validated the expression differences by RT-PCR in additional heterozygous V2424G LCLs from another breast cancer family. We found no consistent cytotoxicity or abrogation of ATM kinase activity after IR in seven heterozygous V2424G LCLs, compared to wild-type LCLs, but did find an increase in the number of chromosomal aberrations. These data suggest that the V2424G missense mutation acts largely as a dominant negative in terms of the associated expression profiles.

Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases.

BACKGROUND: Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse http://variant.imb.uq.edu.au. RESULTS: By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. CONCLUSION: These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways.

Alternate transcription of the Toll-like receptor signaling cascade.

BACKGROUND: Alternate splicing of key signaling molecules in the Toll-like receptor (Tlr) cascade has been shown to dramatically alter the signaling capacity of inflammatory cells, but it is not known how common this mechanism is. We provide transcriptional evidence of widespread alternate splicing in the Toll-like receptor signaling pathway, derived from a systematic analysis of the FANTOM3 mouse data set. Functional annotation of variant proteins was assessed in light of inflammatory signaling in mouse primary macrophages, and the expression of each variant transcript was assessed by splicing arrays. RESULTS: A total of 256 variant transcripts were identified, including novel variants of Tlr4, Ticam1, Tollip, Rac1, Irak1, 2 and 4, Mapk14/p38, Atf2 and Stat1. The expression of variant transcripts was assessed using custom-designed splicing arrays. We functionally tested the expression of Tlr4 transcripts under a range of cytokine conditions via northern and quantitative real-time polymerase chain reaction. The effects of variant Mapk14/p38 protein expression on macrophage survival were demonstrated. CONCLUSION: Members of the Toll-like receptor signaling pathway are highly alternatively spliced, producing a large number of novel proteins with the potential to functionally alter inflammatory outcomes. These variants are expressed in primary mouse macrophages in response to inflammatory mediators such as interferon-gamma and lipopolysaccharide. Our data suggest a surprisingly common role for variant proteins in diversification/repression of inflammatory signaling.

PhosphoregDB: the tissue and sub-cellular distribution of mammalian protein kinases and phosphatases.

BACKGROUND: Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse http://phosphoreg.imb.uq.edu.au that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. RESULTS: The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. CONCLUSION: Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered.

Spatial gene expression in the T-stage mouse metanephros.

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.