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Gene expression profiling of a mouse model of pancreatic islet dysmorphogenesis.

Wilding Crawford L, Tweedie Ables E, Oh YA, Boone B, Levy S, Gannon M - PLoS ONE (2008)

Bottom Line: Despite this success, many of the factors necessary for proper islet morphogenesis and function remain uncharacterized.Genome-wide microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal total pancreas tissue from wild type and Hnf6 transgenic animals.This study provides a unique dataset that can act as a starting point for other investigators to explore the role of the identified genes in pancreatogenesis, islet morphogenesis and mature beta cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

ABSTRACT

Background: In the past decade, several transcription factors critical for pancreas organogenesis have been identified. Despite this success, many of the 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 disruptions in islet morphogenesis, including dysfunctional endocrine cell sorting, increased individual islet size, increased number of peripheral endocrine cell types, and failure of islets to migrate away from the ductal epithelium. The mechanisms whereby maintained Hnf6 causes defects in islet morphogenesis have yet to be elucidated.

Methodology/principal findings: We exploited the dysmorphic islets in Hnf6 transgenic 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 total pancreas tissue from wild type and Hnf6 transgenic animals. Here we report the identification of genes with an altered expression in Hnf6 transgenic animals and highlight factors with potential importance in islet morphogenesis. Importantly, gene products involved in cell adhesion, cell migration, ECM remodeling and proliferation were found to be altered in Hnf6 transgenic pancreata, revealing specific candidates that can now be analyzed directly for their role in these processes during islet development.

Conclusions/significance: This study provides a unique dataset that can act as a starting point for other investigators to explore the role of the identified genes in pancreatogenesis, islet morphogenesis and mature beta cell function.

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Related in: MedlinePlus

RT-PCR validation of microarray results.P1 pancreatic WT (blue) and Hnf6 Tg (purple) RNA was analyzed for the expression of the transcripts indicated. Values are expressed in arbitrary units as a ratio (transcript:tubulin) of the average of individual samples for each genotype. Error bars represent SEM; *p<0.05. n.d., not detectable. For comparison, microarray analysis of these same transcripts revealed the following: OC-1 increased two-fold; Pdx1 no change; Nnat decreased two-fold; Reg2 increased 1.7-fold; Ectodin increased 1.6- to 1.9-fold; Serpina6 increased 4.6-fold; PERK decreased two-fold.
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pone-0001611-g003: RT-PCR validation of microarray results.P1 pancreatic WT (blue) and Hnf6 Tg (purple) RNA was analyzed for the expression of the transcripts indicated. Values are expressed in arbitrary units as a ratio (transcript:tubulin) of the average of individual samples for each genotype. Error bars represent SEM; *p<0.05. n.d., not detectable. For comparison, microarray analysis of these same transcripts revealed the following: OC-1 increased two-fold; Pdx1 no change; Nnat decreased two-fold; Reg2 increased 1.7-fold; Ectodin increased 1.6- to 1.9-fold; Serpina6 increased 4.6-fold; PERK decreased two-fold.

Mentions: In order to validate results obtained using the microarray, we chose several candidate genes for RT-PCR analysis. In these studies, we used individual biological samples to confirm the changes observed with technical replicates used for our microarray studies. For validation purposes, we chose to examine several transcripts that we considered potential factors involved in pancreas development, but that have not been fully characterized (Neuronatin, Reg2, Ectodin, and Serpina6), as well as transcripts that were important for validation of our model (Hnf6, Pdx1). As described, the model of islet dysmorphogenesis used for these studies was islet-specific Hnf6 transgenic over-expression. Levels of Hnf6 (Onecut1) were up-regulated at e18.5 and P1 by two-fold in Hnf6 Tg pancreata on the microarray (Supplementary Material Table S2B, D). Using RT-PCR on individual WT and Hnf6 Tg pancreatic RNA extracts, we were also able to detect an increase in Hnf6 transcript, albeit at slightly lower levels (Figure 3). As Hnf6 has been reported to directly activate Pdx1 early in pancreas development, we predicted we might detect increased levels of Pdx1 in Hnf6 Tg animals on the microarray. No change in Pdx1 expression was detected on the microarray and these results were confirmed with RT-PCR.


Gene expression profiling of a mouse model of pancreatic islet dysmorphogenesis.

Wilding Crawford L, Tweedie Ables E, Oh YA, Boone B, Levy S, Gannon M - PLoS ONE (2008)

RT-PCR validation of microarray results.P1 pancreatic WT (blue) and Hnf6 Tg (purple) RNA was analyzed for the expression of the transcripts indicated. Values are expressed in arbitrary units as a ratio (transcript:tubulin) of the average of individual samples for each genotype. Error bars represent SEM; *p<0.05. n.d., not detectable. For comparison, microarray analysis of these same transcripts revealed the following: OC-1 increased two-fold; Pdx1 no change; Nnat decreased two-fold; Reg2 increased 1.7-fold; Ectodin increased 1.6- to 1.9-fold; Serpina6 increased 4.6-fold; PERK decreased two-fold.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2249940&req=5

pone-0001611-g003: RT-PCR validation of microarray results.P1 pancreatic WT (blue) and Hnf6 Tg (purple) RNA was analyzed for the expression of the transcripts indicated. Values are expressed in arbitrary units as a ratio (transcript:tubulin) of the average of individual samples for each genotype. Error bars represent SEM; *p<0.05. n.d., not detectable. For comparison, microarray analysis of these same transcripts revealed the following: OC-1 increased two-fold; Pdx1 no change; Nnat decreased two-fold; Reg2 increased 1.7-fold; Ectodin increased 1.6- to 1.9-fold; Serpina6 increased 4.6-fold; PERK decreased two-fold.
Mentions: In order to validate results obtained using the microarray, we chose several candidate genes for RT-PCR analysis. In these studies, we used individual biological samples to confirm the changes observed with technical replicates used for our microarray studies. For validation purposes, we chose to examine several transcripts that we considered potential factors involved in pancreas development, but that have not been fully characterized (Neuronatin, Reg2, Ectodin, and Serpina6), as well as transcripts that were important for validation of our model (Hnf6, Pdx1). As described, the model of islet dysmorphogenesis used for these studies was islet-specific Hnf6 transgenic over-expression. Levels of Hnf6 (Onecut1) were up-regulated at e18.5 and P1 by two-fold in Hnf6 Tg pancreata on the microarray (Supplementary Material Table S2B, D). Using RT-PCR on individual WT and Hnf6 Tg pancreatic RNA extracts, we were also able to detect an increase in Hnf6 transcript, albeit at slightly lower levels (Figure 3). As Hnf6 has been reported to directly activate Pdx1 early in pancreas development, we predicted we might detect increased levels of Pdx1 in Hnf6 Tg animals on the microarray. No change in Pdx1 expression was detected on the microarray and these results were confirmed with RT-PCR.

Bottom Line: Despite this success, many of the factors necessary for proper islet morphogenesis and function remain uncharacterized.Genome-wide microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal total pancreas tissue from wild type and Hnf6 transgenic animals.This study provides a unique dataset that can act as a starting point for other investigators to explore the role of the identified genes in pancreatogenesis, islet morphogenesis and mature beta cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

ABSTRACT

Background: In the past decade, several transcription factors critical for pancreas organogenesis have been identified. Despite this success, many of the 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 disruptions in islet morphogenesis, including dysfunctional endocrine cell sorting, increased individual islet size, increased number of peripheral endocrine cell types, and failure of islets to migrate away from the ductal epithelium. The mechanisms whereby maintained Hnf6 causes defects in islet morphogenesis have yet to be elucidated.

Methodology/principal findings: We exploited the dysmorphic islets in Hnf6 transgenic 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 total pancreas tissue from wild type and Hnf6 transgenic animals. Here we report the identification of genes with an altered expression in Hnf6 transgenic animals and highlight factors with potential importance in islet morphogenesis. Importantly, gene products involved in cell adhesion, cell migration, ECM remodeling and proliferation were found to be altered in Hnf6 transgenic pancreata, revealing specific candidates that can now be analyzed directly for their role in these processes during islet development.

Conclusions/significance: This study provides a unique dataset that can act as a starting point for other investigators to explore the role of the identified genes in pancreatogenesis, islet morphogenesis and mature beta cell function.

Show MeSH
Related in: MedlinePlus