Project description:The v-erbA oncogene belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid (RA). Nuclear receptors bind to specific DNA sequences in the promoter region of target genes and v-erbA is known to exert a dominant negative effect on the activity of the RARs. The repressor activity of v-erbA has been linked to the development of hepatocellular carcinoma (HCC) in a mouse model. We have used microarray analysis to identify genes differentially expressed in hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA. We have found that v-erbA can affect expression of RA-responsive genes. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC.
Project description:Retinoic acid (RA) signaling regulates a variety of developmental processes through controlling the expression of numerous genes. Here, we have identified and characterized RA-responsive genes in mouse kidney development. Analysis of isolated embryonic kidneys cultured in the presence and absence of RA identified 33 candidates of RA-responsive genes. Most of these candidate genes were down-regulated by treatment with the RA receptor antagonist. Many of them have potential binding sites for Elf5, one of the RA-responsive genes, in their promoter region. Whole-mount in situ hybridization demonstrated that specific expression of Elf5 in the ureteric trunk depends on RA. RA-dependent expression in the ureteric trunk was also demonstrated for the sodium channel subunit Scnn1b, which has been shown to be the marker gene of the collecting duct. In contrast, the expression of Ecm1, Tnfsf13b and IL-33 was detected in the stromal mesenchymal cells. Both Tnfsf13b and IL-33 were previously shown to cause NF-κB activation. We have demonstrated that the inhibition of NF-κB signaling with specific inhibitors suppresses branching morphogenesis of the ureteric bud. Our study thus identifies and characterizes RA-dependent upregulated genes in kidney development, and suggests an involvement of NF-κB signaling in the branching morphogenesis.
Project description:To explore potential impact of Aldh1a1 deficiency in the retinoic acid signaling in the lung, gene expression levels were compared between wildtype and Aldh1a1 deficient lung tissues. The expression levels of retinoic acid responsive genes were very similar between wildtype and Aldh1a1 deficient lungs, suggesting that Aldh1a1 deficiency has minimal impact on retinoic acid signaling.
Project description:The v-erbA oncogene belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid (RA). Nuclear receptors bind to specific DNA sequences in the promoter region of target genes and v-erbA is known to exert a dominant negative effect on the activity of the RARs. The repressor activity of v-erbA has been linked to the development of hepatocellular carcinoma (HCC) in a mouse model. We have used microarray analysis to identify genes differentially expressed in hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA. We have found that v-erbA can affect expression of RA-responsive genes. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC. Experiment Overall Design: AML12 control cells and v-erbA-transfected AML12 cells were exposed to 1 µM RA for 3h or 24h. Using microarray analysis, we compared gene expression in the presence and absence of v-erbA and identified RA-regulated genes differentially expressed in the presence of v-erbA.
Project description:Retinoic acid (RA) signaling regulates a variety of developmental processes through controlling the expression of numerous genes. Here, we have identified and characterized RA-responsive genes in mouse kidney development. Analysis of isolated embryonic kidneys cultured in the presence and absence of RA identified 33 candidates of RA-responsive genes. Most of these candidate genes were down-regulated by treatment with the RA receptor antagonist. Many of them have potential binding sites for Elf5, one of the RA-responsive genes, in their promoter region. Whole-mount in situ hybridization demonstrated that specific expression of Elf5 in the ureteric trunk depends on RA. RA-dependent expression in the ureteric trunk was also demonstrated for the sodium channel subunit Scnn1b, which has been shown to be the marker gene of the collecting duct. In contrast, the expression of Ecm1, Tnfsf13b and IL-33 was detected in the stromal mesenchymal cells. Both Tnfsf13b and IL-33 were previously shown to cause NF-?B activation. We have demonstrated that the inhibition of NF-?B signaling with specific inhibitors suppresses branching morphogenesis of the ureteric bud. Our study thus identifies and characterizes RA-dependent upregulated genes in kidney development, and suggests an involvement of NF-?B signaling in the branching morphogenesis. To obtain genome-wide gene expression profiles of kidneys cultured in 10% FBS DMEM, serum-free DMEM, and serum-free DMEM with RA, we performed microarray analysis on RNA prepared from these three samples. Total RNA was prepared using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instruction. Synthesis of cDNA, in vitro transcription and biotin labeling cRNA, and hybridization to GeneChip Mouse Gene 1.0 ST array (Affymetrix) were performed according to Affymetrix protocols.
Project description:Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we employed microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 upregulated genes (92% of which were validated) including known RA responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). 67 validated downregulated genes were identified including known RA responsive genes and anterior marker genes. The expression patterns of selected upregulated genes (n = 45) were examined at neurula stages using whole mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways and their further characterization will expand our understanding of RA signaling during development. Keywords = retinoid Keywords = microarray Keywords = RAR Keywords = neurula Keywords = anteroposterior patterning Keywords = and organogenesis