PMC:1858683 / 23618-29959 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/1858683","sourcedb":"PMC","sourceid":"1858683","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1858683","text":"Microarray analysis of Pygo1/Pygo2 null kidneys\nTo further examine possible disturbance of gene expression in the Pygo1/Pygo2 mutant kidneys, we used microarrays to perform a global analysis of gene expression changes. Whereas the BAT-gal transgene reporter monitors the response of one promoter to Wnt signaling, microarrays can be used to follow expression changes of all genes, including all known Wnt targets. E18.5 wild-type and Pygo1-/-/Pygo2-/- kidneys were examined in biological triplicate. In total, 45 genes were identified as significantly changed, using a relatively low stringency screen of the data, with a p-value cutoff of \u003c0.05, and fold change \u003e2 (Table 3). Notably, both Pygo1 and Pygo2 were identified as downregulated (- 5.3-fold and - 3.9-fold, respectively) in the mutant kidneys. Other genes with significant decreases in mutant kidneys included Cxcl13 (- 2.3-fold), Slc5a2 (- 2.8-fold), and Slco1a4 (- 2.1-fold). Slc5a2 is expressed in the proximal tubules of the adult nephron and has been implicated in autosomal recessive renal glucosuria, characterized by loss of glucose uptake by the nephron [23]. The organic anion transporter, Slco1a4, is also strongly expressed in the tubules of the adult kidney [24]. These results suggest that the Pygo1/Pygo2 genes might play a role in nephron maturation and subsequent function.\nTable 3 Genes differentially expressed (p \u003c 0.05, two-fold change or greater) in the Pygo1/Pygo2 null E18.5 kidney normalized to wild-type samples.\nGene symbol Gene name Pygo1/2 null average fold change\nC920006O11Rik Hypothetical protein 7.1\nTia1 Cytotoxic granule-assoc RNA binding 1 6.6\nAkr1e1 Aldo-keto reductase family 1, E1 5.5\n5830417I10Rik Hypothetical protein 3.8\nXM_193262 Protein 40kD (LOC269251) mRNA. 3.8\nPicalm Pl-binding clathrin assembly protein 3.1\nPck1 PEP carboxykinase 1, cytosolic 2.9\nKlk5 Kallikrein 5 2.8\nKlk6 Kallikrein 6 2.7\nAK051496 4933409K07Rik protein 2.5\nTimeless Timeless homolog 2.4\nKlk27 Kallikrein 27 2.4\nGsta2 Glutathione S-transferase, alpha 1 2.4\nTia1 cytotoxic granule-assoc RNA binding 1 2.3\nAK049070 Hypothetical protein 2.3\nBC051083 Cisplatin resistance associated 2.3\n1500015O10Rik Esophageal cancer related gene 4 2.3\nKlk27 Kallikrein 27 2.2\nNpy Neuropeptide Y 2.2\nRen2 Renin 2 2.2\nGsta2 Glutathione S-transferase, alpha 2 2.1\nCol8a1 Procollagen, type VIII, alpha 1 2.1\nCol8a1 Procollagen, type VIII, alpha 1 2.1\nG6pc Glucose-6-phosphatase, catalytic 2.1\nRgs4 Regulator of G-protein signaling 4 2.0\nXM_358675 Hypothetical protein 2.0\nCtse Cathepsin E preproprotein - 2.0\nMasp2 Mannan-binding lectin serine protease 2 - 2.0\nSlco1a4 Solute carrier organic anion transporter 1a4 - 2.1\nCyp2e1 Cytochrome P450 2E1 - 2.1\nSah SA hypertension-associated homolog - 2.1\nAldh1a7 Aldehyde dehydrogenase 1A7 - 2.2\n1200006F02Rik Hypothetical protein - 2.3\n2600001B17Rik Hypothetical protein - 2.3\nD630004N19Rik Hypothetical protein - 2.3\nCxcl13 Chemokine (C-X-C motif) ligand 13 - 2.3\nHpgd Hydroxyprostaglandin dehydrogenase 15 - 2.4\nLum Lumican - 2.5\nPvalb Parvalbumin - 2.5\nCml4 N-acetyltransferase Camello 4 - 2.7\nSlc5a2 Solute carrier 5 (sodium/glucose) 2 - 2.8\nPygo2 Pygopus 2 - 3.9\nPygo1 Pygopus 1 - 5.3\n2610305D13Rik Hypothetical protein - 5.6\nCsrp1 Cysteine and glycine-rich protein 1 - 7.1 Noteworthy genes upregulated in the Pygo1/Pygo2 null kidney included Klk5 (2.8-fold), Klk6 (2.7-fold), Ren2 (2.2-fold), and Timeless (2.4-fold). Klk5 and Klk6 are members of the kallikrein family of trypsin-like serine proteases. Kallikreins have diverse functions in cancer, tissue remodeling, and regulation of blood pressure [25]. Ren2, a homolog of the endopeptidase Renin 1, activates the renin-angiotensin system, increasing blood pressure [26]. Disruption of the renin-angiotensin system during development results in congenital abnormalities of the ureter and collecting-duct system [27]. Timeless, a transcription factor involved in the regulation of circadian rhythms, and expressed in the branching ureteric tips, has also been shown to regulate ureteric branching morphogenesis [28].\nWe were interested in the possible altered expression of previously known Wnt targets in the Pygo1-/-/Pygo2-/- mutant kidneys. A list of known Wnt targets was compiled from . Both Pygo1 and Pygo2 probe sets were included as references to illustrate significant down regulation. Remarkably, the known Wnt target genes showed very few differences in expression between wild-type and mutant kidneys (Figure 8). Only the expression of Ccnd1 encoding cyclin D1 [29], and Wisp1 [30] were significantly changed, with expression level changes of \u003c1.5-fold in Pygo1/Pygo2 null kidneys. These results indicate an absence of dramatic changes in expression of previously known Wnt signaling target genes in the Pygo1/Pygo2 mutant kidneys.\nFigure 8 Gene expression changes of common Wnt signaling targets in the E18.5 Pygo1/Pygo2 null kidney. Microarray analysis was performed in triplicate on wild-type and Pygo1/2 compound null E18.5 kidneys. Possible Wnt targets were selected from those compiled at [39]. An initial gene list of 82 Wnt targets was created and then reduced to a total of 40 genes using an expression level restriction requiring the raw expression intensity to be \u003e100 in at least 3 samples. Pygo1 and Pygo2 probes were included to demonstrate significant changes in expression levels. We used quantitative real-time PCR, with independent biological samples, to validate the microarray results. Nine genes were tested, and for seven we did observe a significant fold change in the same direction predicted by the microarray results (Table 4). It is not surprising that two of the nine genes could not be validated, considering the relatively low stringency used in screening the microarray data.\nTable 4 Validation of gene expression changes in the E18.5 Pygo1/Pygo2 null kidneys normalized to E18.5 wild-type kidneys.\nGene symbol Microarray fold change QPCR fold change (SD) Validation\nAldh1a7 - 2.2 1.1 (0.11) No\nCol8a1 2.1 2.9 (0.78) Yes\nCsrp1 - 7.1 1.2 (0.02) No\nKlk5 2.8 5.8 (0.74) Yes\nPygo2 - 3.9 - 3.7 (0.04) Yes\nPicalm 3.1 2.2 (0.24) Yes\nRen2 2.2 6.1 (0.36) Yes\nTia1 6.6 and 2.3 Amplification not detected in wild-type samples 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