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Identification of transcripts with enriched expression in the developing and adult pancreas.

Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Beach M, Hoodless PA, Jones SJ, Marra MA, Helgason CD - Genome Biol. (2008)

Bottom Line: Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development.Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Endocrinology, BC Cancer Research Center, West 10th Ave, Vancouver, BC V5Z 1L3, Canada. bhoffman@bccrc.ca

ABSTRACT

Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.

Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.

Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

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Related in: MedlinePlus

A cascade of transcription factors expressed in endocrine cell development. Tags that mapped unambiguously to transcription factors and met our count and specificity thresholds were identified and their normalized counts obtained in each of the SAGE libraries that represent the development of the endocrine lineage. (a) A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown. Tags are organized by the order of their expression in the libraries. The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); adult isolated islets (Islets). (b) A transcription factor network in β-cell development. The network was generated as described in the materials and methods using Biotapestry to visualize the network [65]. Ins1 and Ins2 are shown as the final products expressed in mature β-cells. Literature reported interactions [8,41] are included with arrow heads indicating positive regulation and perpendicular lines repression. Interactions in pancreatic progenitors (PP), endocrine progenitors (EP) and mature β-cells are shown. (c) The fold enrichment of predicted Foxa2 binding sites, as detected by qPCR, in the promoters of Pdx1, Myt1, Myt3, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Foxa2 site (NegF1 and NegF2) in ChIP experiments using an anti-Foxa2 antibody compared to IgG. (d) The fold enrichment of predicted Pdx1 binding sites, as detected by qPCR, in the promoters of Foxa2, Ins1, Myt1, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Pdx1 site (NegP1 and NegP2) in ChIP experiments using an anti-Pdx1 antibody compared to IgG. The data shown are averages of the results obtained from three separate ChIP experiments each with duplicate reactions. Error bars indicate the standard deviation of the averaged replicates.
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Figure 7: A cascade of transcription factors expressed in endocrine cell development. Tags that mapped unambiguously to transcription factors and met our count and specificity thresholds were identified and their normalized counts obtained in each of the SAGE libraries that represent the development of the endocrine lineage. (a) A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown. Tags are organized by the order of their expression in the libraries. The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); adult isolated islets (Islets). (b) A transcription factor network in β-cell development. The network was generated as described in the materials and methods using Biotapestry to visualize the network [65]. Ins1 and Ins2 are shown as the final products expressed in mature β-cells. Literature reported interactions [8,41] are included with arrow heads indicating positive regulation and perpendicular lines repression. Interactions in pancreatic progenitors (PP), endocrine progenitors (EP) and mature β-cells are shown. (c) The fold enrichment of predicted Foxa2 binding sites, as detected by qPCR, in the promoters of Pdx1, Myt1, Myt3, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Foxa2 site (NegF1 and NegF2) in ChIP experiments using an anti-Foxa2 antibody compared to IgG. (d) The fold enrichment of predicted Pdx1 binding sites, as detected by qPCR, in the promoters of Foxa2, Ins1, Myt1, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Pdx1 site (NegP1 and NegP2) in ChIP experiments using an anti-Pdx1 antibody compared to IgG. The data shown are averages of the results obtained from three separate ChIP experiments each with duplicate reactions. Error bars indicate the standard deviation of the averaged replicates.

Mentions: Transcriptional regulators, in concert with signaling factors, provide the genetic instructions that drive endocrine pancreas development. Based on our analyses, genes in the endocrine lineage (that is, from pancreatic progenitor cells through endocrine progenitors to adult islet cells) can be identified based on their presence in clusters 1, 2, 4, 5, 8 and 13. We identified 58 tags for transcriptional regulators in these clusters representing 43 different factors. Eliminating factors only expressed in one library, and for which we could not find additional support for their expression, left 38 different factors for which there is good evidence of their expression in the endocrine pancreas lineage. Eight of these genes were represented by multiple tag types, with four separate tags mapping to Neurod1 and Isl1. Figure 7a shows a heatmap of the expression of these factors in endocrine cell development as detected in our SAGE data. Many of these factors have well-established roles in pancreas development, including Ngn3, Pax4, Nkx2-2, Pdx1, Isl1, and Neurod1. However, several factors with uncharacterized roles were also identified, including: Tcf12, Zfp326 and Meox1, which were most abundant in Pdx1 EGFP+ cells; Zfp446, Rnf6, and Son, which were most abundant in Neurog3 EGFP+ cells; and Nr1d1, Myt3, and Bcl6b, which were most abundant in islet cells.


Identification of transcripts with enriched expression in the developing and adult pancreas.

Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Beach M, Hoodless PA, Jones SJ, Marra MA, Helgason CD - Genome Biol. (2008)

A cascade of transcription factors expressed in endocrine cell development. Tags that mapped unambiguously to transcription factors and met our count and specificity thresholds were identified and their normalized counts obtained in each of the SAGE libraries that represent the development of the endocrine lineage. (a) A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown. Tags are organized by the order of their expression in the libraries. The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); adult isolated islets (Islets). (b) A transcription factor network in β-cell development. The network was generated as described in the materials and methods using Biotapestry to visualize the network [65]. Ins1 and Ins2 are shown as the final products expressed in mature β-cells. Literature reported interactions [8,41] are included with arrow heads indicating positive regulation and perpendicular lines repression. Interactions in pancreatic progenitors (PP), endocrine progenitors (EP) and mature β-cells are shown. (c) The fold enrichment of predicted Foxa2 binding sites, as detected by qPCR, in the promoters of Pdx1, Myt1, Myt3, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Foxa2 site (NegF1 and NegF2) in ChIP experiments using an anti-Foxa2 antibody compared to IgG. (d) The fold enrichment of predicted Pdx1 binding sites, as detected by qPCR, in the promoters of Foxa2, Ins1, Myt1, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Pdx1 site (NegP1 and NegP2) in ChIP experiments using an anti-Pdx1 antibody compared to IgG. The data shown are averages of the results obtained from three separate ChIP experiments each with duplicate reactions. Error bars indicate the standard deviation of the averaged replicates.
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Figure 7: A cascade of transcription factors expressed in endocrine cell development. Tags that mapped unambiguously to transcription factors and met our count and specificity thresholds were identified and their normalized counts obtained in each of the SAGE libraries that represent the development of the endocrine lineage. (a) A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown. Tags are organized by the order of their expression in the libraries. The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); adult isolated islets (Islets). (b) A transcription factor network in β-cell development. The network was generated as described in the materials and methods using Biotapestry to visualize the network [65]. Ins1 and Ins2 are shown as the final products expressed in mature β-cells. Literature reported interactions [8,41] are included with arrow heads indicating positive regulation and perpendicular lines repression. Interactions in pancreatic progenitors (PP), endocrine progenitors (EP) and mature β-cells are shown. (c) The fold enrichment of predicted Foxa2 binding sites, as detected by qPCR, in the promoters of Pdx1, Myt1, Myt3, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Foxa2 site (NegF1 and NegF2) in ChIP experiments using an anti-Foxa2 antibody compared to IgG. (d) The fold enrichment of predicted Pdx1 binding sites, as detected by qPCR, in the promoters of Foxa2, Ins1, Myt1, Neurod1, Nkx2-2, Nkx6-1, and of two sites not predicted to contain a Pdx1 site (NegP1 and NegP2) in ChIP experiments using an anti-Pdx1 antibody compared to IgG. The data shown are averages of the results obtained from three separate ChIP experiments each with duplicate reactions. Error bars indicate the standard deviation of the averaged replicates.
Mentions: Transcriptional regulators, in concert with signaling factors, provide the genetic instructions that drive endocrine pancreas development. Based on our analyses, genes in the endocrine lineage (that is, from pancreatic progenitor cells through endocrine progenitors to adult islet cells) can be identified based on their presence in clusters 1, 2, 4, 5, 8 and 13. We identified 58 tags for transcriptional regulators in these clusters representing 43 different factors. Eliminating factors only expressed in one library, and for which we could not find additional support for their expression, left 38 different factors for which there is good evidence of their expression in the endocrine pancreas lineage. Eight of these genes were represented by multiple tag types, with four separate tags mapping to Neurod1 and Isl1. Figure 7a shows a heatmap of the expression of these factors in endocrine cell development as detected in our SAGE data. Many of these factors have well-established roles in pancreas development, including Ngn3, Pax4, Nkx2-2, Pdx1, Isl1, and Neurod1. However, several factors with uncharacterized roles were also identified, including: Tcf12, Zfp326 and Meox1, which were most abundant in Pdx1 EGFP+ cells; Zfp446, Rnf6, and Son, which were most abundant in Neurog3 EGFP+ cells; and Nr1d1, Myt3, and Bcl6b, which were most abundant in islet cells.

Bottom Line: Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development.Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Endocrinology, BC Cancer Research Center, West 10th Ave, Vancouver, BC V5Z 1L3, Canada. bhoffman@bccrc.ca

ABSTRACT

Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.

Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.

Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

Show MeSH
Related in: MedlinePlus