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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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Immunofluorescent and western blot validation of microarray results for Hnf6 transcriptional targets.No changes in Pdx1 expression (red) were observed by co-immunohistochemistry in e18.5 WT (A) and Hnf6 Tg (B) embryonic pancreata (glucagon shown in green). In contrast, Increased numbers of Ngn3+ cells (green) were observed within the pancreatic epithelium (outlined) at e15.5 (glucagon+ cells shown in red) in HNF6 Tg pancreata (C) as compared to WT pancreata (D). P1 pancreatic extracts (E) were probed for the expression of the proteins indicated. Values are expressed as a ratio (WT:Hnf6 Tg). Two representative samples for each genotype are shown.
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pone-0001611-g005: Immunofluorescent and western blot validation of microarray results for Hnf6 transcriptional targets.No changes in Pdx1 expression (red) were observed by co-immunohistochemistry in e18.5 WT (A) and Hnf6 Tg (B) embryonic pancreata (glucagon shown in green). In contrast, Increased numbers of Ngn3+ cells (green) were observed within the pancreatic epithelium (outlined) at e15.5 (glucagon+ cells shown in red) in HNF6 Tg pancreata (C) as compared to WT pancreata (D). P1 pancreatic extracts (E) were probed for the expression of the proteins indicated. Values are expressed as a ratio (WT:Hnf6 Tg). Two representative samples for each genotype are shown.

Mentions: Approximately 20% of all annotated transcripts that showed altered expression on the microarray were categorized as nucleic acid binding proteins (Figure 4). This is representative of the complete annotated probeset, in which 19.6% of all transcripts encode nucleic acid binding proteins. One transcript of interest, the pro-endocrine transcription factor Ngn3 (Neurog3), was up-regulated two-fold at e18.5 by microarray analysis (Supplementary Material Tables S1B, S2B). Lineage tracing [42] and global deletion analyses [43] have shown that Ngn3 is required for the differentiation of all endocrine cells in the pancreas, and as such is one of the earliest transcription factors that specifically marks the endocrine population prior to cell type-specific hormone expression [44]. Previous research has shown that Ngn3 is a direct target of Hnf6 transcriptional activity; global Hnf6−/− mice have a dramatic down-regulation of Ngn3+ cells [29]. Furthermore, over-expression of Ngn3 within the Pdx1+ domain results in an expansion of the endocrine population, specifically in glucagon-producing cells [44]. Thus, the increased numbers of glucagon+ cells we observed in Hnf6 Tg pancreata at e15.5, e18.5, and P1 (Figures 1C and 5B, E) may be due, in part, to increased expression of Ngn3. We have used both western blot and immunohistochemistry analyses to validate the up-regulation of Ngn3 transcripts in Hnf6 Tg pancreata (Figure 5). These experiments show that the number of Ngn3+ cells at e15.5 and the amount of Ngn3 protein at P1 (1.2 fold increase in Tg) is increased in Hnf6 Tg pancreata (Figures 5C–E). This data correlates well with the up-regulation of Ngn3 in the e18.5 microarray data set (Supplementary Material Table S2B). Thus, over-expression of Hnf6 specifically in the endocrine lineage results either in an increase in the absolute number of Ngn3+ endocrine progenitor cells, or increases the duration of NGN3 expression in these cells. Regardless of the mechanism, the effects of HNF6 over-expression on Ngn3 expression are transient, as microarray analysis at P1 failed to show an increase in Ngn3 expression (Supplementary Material Table S2D).


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)

Immunofluorescent and western blot validation of microarray results for Hnf6 transcriptional targets.No changes in Pdx1 expression (red) were observed by co-immunohistochemistry in e18.5 WT (A) and Hnf6 Tg (B) embryonic pancreata (glucagon shown in green). In contrast, Increased numbers of Ngn3+ cells (green) were observed within the pancreatic epithelium (outlined) at e15.5 (glucagon+ cells shown in red) in HNF6 Tg pancreata (C) as compared to WT pancreata (D). P1 pancreatic extracts (E) were probed for the expression of the proteins indicated. Values are expressed as a ratio (WT:Hnf6 Tg). Two representative samples for each genotype are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2249940&req=5

pone-0001611-g005: Immunofluorescent and western blot validation of microarray results for Hnf6 transcriptional targets.No changes in Pdx1 expression (red) were observed by co-immunohistochemistry in e18.5 WT (A) and Hnf6 Tg (B) embryonic pancreata (glucagon shown in green). In contrast, Increased numbers of Ngn3+ cells (green) were observed within the pancreatic epithelium (outlined) at e15.5 (glucagon+ cells shown in red) in HNF6 Tg pancreata (C) as compared to WT pancreata (D). P1 pancreatic extracts (E) were probed for the expression of the proteins indicated. Values are expressed as a ratio (WT:Hnf6 Tg). Two representative samples for each genotype are shown.
Mentions: Approximately 20% of all annotated transcripts that showed altered expression on the microarray were categorized as nucleic acid binding proteins (Figure 4). This is representative of the complete annotated probeset, in which 19.6% of all transcripts encode nucleic acid binding proteins. One transcript of interest, the pro-endocrine transcription factor Ngn3 (Neurog3), was up-regulated two-fold at e18.5 by microarray analysis (Supplementary Material Tables S1B, S2B). Lineage tracing [42] and global deletion analyses [43] have shown that Ngn3 is required for the differentiation of all endocrine cells in the pancreas, and as such is one of the earliest transcription factors that specifically marks the endocrine population prior to cell type-specific hormone expression [44]. Previous research has shown that Ngn3 is a direct target of Hnf6 transcriptional activity; global Hnf6−/− mice have a dramatic down-regulation of Ngn3+ cells [29]. Furthermore, over-expression of Ngn3 within the Pdx1+ domain results in an expansion of the endocrine population, specifically in glucagon-producing cells [44]. Thus, the increased numbers of glucagon+ cells we observed in Hnf6 Tg pancreata at e15.5, e18.5, and P1 (Figures 1C and 5B, E) may be due, in part, to increased expression of Ngn3. We have used both western blot and immunohistochemistry analyses to validate the up-regulation of Ngn3 transcripts in Hnf6 Tg pancreata (Figure 5). These experiments show that the number of Ngn3+ cells at e15.5 and the amount of Ngn3 protein at P1 (1.2 fold increase in Tg) is increased in Hnf6 Tg pancreata (Figures 5C–E). This data correlates well with the up-regulation of Ngn3 in the e18.5 microarray data set (Supplementary Material Table S2B). Thus, over-expression of Hnf6 specifically in the endocrine lineage results either in an increase in the absolute number of Ngn3+ endocrine progenitor cells, or increases the duration of NGN3 expression in these cells. Regardless of the mechanism, the effects of HNF6 over-expression on Ngn3 expression are transient, as microarray analysis at P1 failed to show an increase in Ngn3 expression (Supplementary Material Table S2D).

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