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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

Heatmap of SAGE tag counts for genes with known expression profiles in pancreas development. Tags for genes with well characterized expression profiles in pancreas development were identified and their normalized counts obtained in each of the ten SAGE libraries created. A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown based on the counts of the tags per hundred thousand (TPH). SAGE tags used include: TACACGTTCTGACAACT (Nkx2-2); AAGTGGAAAAAAGAGGA (Pdx1); TAGTTTTAACAGAAAAC (Foxa2); ACCTTCACACCAAACAT (Hnf4a); AATGCAGAGGAGGACTC (Neurod1); CAGGGTTTCTGAGCTTC (Neurog3); TCATTTGACTTTTTTTT (Isl1); GATTTAAGAGTTTTATC (Pax6); CAGCAGGACGGACTCAG (Pax4); CAGTCCATCAACGACGC (Ptf1a); AGAAACAGCAGGGCCTG (Bhlhb8); GACCACACTGTCAAACA (Cpa1); CCCTGGGTTCAGGAGAT (Ctrb1); TTGCGCTTCCTGGTGTT (Ela1); ACCACCTGGTAACCGTA (Gcg); GCCGGGCCCTGGGGAAG (Ghrl); CTAAGAATTGCTTTAAA (Iapp); GCCCTGTTGGTGCACTT (Ins1); TCCCGCCGTGAAGTGGA (Ins2). The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP- TS20 (N- TS20); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); whole pancreas TS22 (WTS22); whole pancreas TS26 (WTS26); adult isolated ducts (Ducts); adult isolated islets (Islets).
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Figure 1: Heatmap of SAGE tag counts for genes with known expression profiles in pancreas development. Tags for genes with well characterized expression profiles in pancreas development were identified and their normalized counts obtained in each of the ten SAGE libraries created. A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown based on the counts of the tags per hundred thousand (TPH). SAGE tags used include: TACACGTTCTGACAACT (Nkx2-2); AAGTGGAAAAAAGAGGA (Pdx1); TAGTTTTAACAGAAAAC (Foxa2); ACCTTCACACCAAACAT (Hnf4a); AATGCAGAGGAGGACTC (Neurod1); CAGGGTTTCTGAGCTTC (Neurog3); TCATTTGACTTTTTTTT (Isl1); GATTTAAGAGTTTTATC (Pax6); CAGCAGGACGGACTCAG (Pax4); CAGTCCATCAACGACGC (Ptf1a); AGAAACAGCAGGGCCTG (Bhlhb8); GACCACACTGTCAAACA (Cpa1); CCCTGGGTTCAGGAGAT (Ctrb1); TTGCGCTTCCTGGTGTT (Ela1); ACCACCTGGTAACCGTA (Gcg); GCCGGGCCCTGGGGAAG (Ghrl); CTAAGAATTGCTTTAAA (Iapp); GCCCTGTTGGTGCACTT (Ins1); TCCCGCCGTGAAGTGGA (Ins2). The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP- TS20 (N- TS20); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); whole pancreas TS22 (WTS22); whole pancreas TS26 (WTS26); adult isolated ducts (Ducts); adult isolated islets (Islets).

Mentions: To confirm that the libraries accurately represent the cell types intended (Table 1), we assessed the distribution of tags in the libraries for genes with well-characterized expression profiles in pancreas development. Figure 1 shows that transcription factors expressed in pancreas progenitor epithelial cells, such as Pdx1 and Nkx2-2, can be found in our TS17-TS19 Pdx1 EGFP+ libraries. Tags for these genes were also found frequently in the Neurog3 EGFP+ libraries. This is in agreement with the known expression of these factors. For example, Pdx1 is expressed in essentially all pancreas epithelial cells prior to the secondary transition while its expression after the secondary transition is abundant only in β-cells and β-cell precursors [8]. Prior to the secondary transition Neurog3 expression is quite low; however, at the start of the secondary transition its expression increases dramatically [31] and is subsequently lost quickly thereafter. This is precisely what we see in our data - low Neurog3 levels in the Pdx1 EGFP+ libraries, high expression in the Neurog3 EGFP+ libraries and diminishing expression in the TS22 and TS26 whole pancreas libraries, with no expression in the Neurog3 EGFP- or the adult islet or duct libraries. Neurod1, Isl1, Pax6 and Pax4 expression occurs subsequent to Neurog3, but unlike Neurog3 their expression is maintained in endocrine cell types [8]. In our data it is clear that the expression of all of these genes is most abundant in the Neurog3 EGFP+ libraries, or the islet library, as would be predicted. Ptf1a and Bhlhb8 (Mist1) are two transcription factors known to drive exocrine cell development. Ptf1a was found only in the TS22 whole pancreas library, and while low levels of Bhlhb8 were noted in the TS22 Neurog3 EGFP+ library, much higher levels were found in the duct cell library. Markers of mature exocrine cells showed peak expression in the TS26 whole pancreas or adult duct libraries, with moderate expression also in the islet library, suggesting a low level of exocrine cell contamination in this library. Glucagon expression peaked in the Neurog3 EGFP+ libraries, which is not surprising as Glucagon-positive cells are relatively abundant at these time points compared to in the adult islet. Iapp, Ins1 and Ins2 were all most abundant in the islet library, as was expected. The expression of these genes was also noted in the duct library, suggesting some level of islet cell contamination in this library. In sum, the expression profiles of these selected markers in our data match predictions based on their known expression profiles, indicating that our libraries accurately reflect the cell types and stages intended.


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)

Heatmap of SAGE tag counts for genes with known expression profiles in pancreas development. Tags for genes with well characterized expression profiles in pancreas development were identified and their normalized counts obtained in each of the ten SAGE libraries created. A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown based on the counts of the tags per hundred thousand (TPH). SAGE tags used include: TACACGTTCTGACAACT (Nkx2-2); AAGTGGAAAAAAGAGGA (Pdx1); TAGTTTTAACAGAAAAC (Foxa2); ACCTTCACACCAAACAT (Hnf4a); AATGCAGAGGAGGACTC (Neurod1); CAGGGTTTCTGAGCTTC (Neurog3); TCATTTGACTTTTTTTT (Isl1); GATTTAAGAGTTTTATC (Pax6); CAGCAGGACGGACTCAG (Pax4); CAGTCCATCAACGACGC (Ptf1a); AGAAACAGCAGGGCCTG (Bhlhb8); GACCACACTGTCAAACA (Cpa1); CCCTGGGTTCAGGAGAT (Ctrb1); TTGCGCTTCCTGGTGTT (Ela1); ACCACCTGGTAACCGTA (Gcg); GCCGGGCCCTGGGGAAG (Ghrl); CTAAGAATTGCTTTAAA (Iapp); GCCCTGTTGGTGCACTT (Ins1); TCCCGCCGTGAAGTGGA (Ins2). The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP- TS20 (N- TS20); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); whole pancreas TS22 (WTS22); whole pancreas TS26 (WTS26); adult isolated ducts (Ducts); adult isolated islets (Islets).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481431&req=5

Figure 1: Heatmap of SAGE tag counts for genes with known expression profiles in pancreas development. Tags for genes with well characterized expression profiles in pancreas development were identified and their normalized counts obtained in each of the ten SAGE libraries created. A heatmap, generated using the multi-experiment viewer as described in the Materials and methods, of these results is shown based on the counts of the tags per hundred thousand (TPH). SAGE tags used include: TACACGTTCTGACAACT (Nkx2-2); AAGTGGAAAAAAGAGGA (Pdx1); TAGTTTTAACAGAAAAC (Foxa2); ACCTTCACACCAAACAT (Hnf4a); AATGCAGAGGAGGACTC (Neurod1); CAGGGTTTCTGAGCTTC (Neurog3); TCATTTGACTTTTTTTT (Isl1); GATTTAAGAGTTTTATC (Pax6); CAGCAGGACGGACTCAG (Pax4); CAGTCCATCAACGACGC (Ptf1a); AGAAACAGCAGGGCCTG (Bhlhb8); GACCACACTGTCAAACA (Cpa1); CCCTGGGTTCAGGAGAT (Ctrb1); TTGCGCTTCCTGGTGTT (Ela1); ACCACCTGGTAACCGTA (Gcg); GCCGGGCCCTGGGGAAG (Ghrl); CTAAGAATTGCTTTAAA (Iapp); GCCCTGTTGGTGCACTT (Ins1); TCCCGCCGTGAAGTGGA (Ins2). The libraries shown include: Pdx1 EGFP+ TS17 (P+ TS17); Pdx1 EGFP+ TS19 (P+ TS19); Neurog3 EGFP- TS20 (N- TS20); Neurog3 EGFP+ TS20 (N+ TS20); Neurog3 EGFP+ TS21 (N+ TS21); Neurog3 EGFP+ TS22 (N+ TS22); whole pancreas TS22 (WTS22); whole pancreas TS26 (WTS26); adult isolated ducts (Ducts); adult isolated islets (Islets).
Mentions: To confirm that the libraries accurately represent the cell types intended (Table 1), we assessed the distribution of tags in the libraries for genes with well-characterized expression profiles in pancreas development. Figure 1 shows that transcription factors expressed in pancreas progenitor epithelial cells, such as Pdx1 and Nkx2-2, can be found in our TS17-TS19 Pdx1 EGFP+ libraries. Tags for these genes were also found frequently in the Neurog3 EGFP+ libraries. This is in agreement with the known expression of these factors. For example, Pdx1 is expressed in essentially all pancreas epithelial cells prior to the secondary transition while its expression after the secondary transition is abundant only in β-cells and β-cell precursors [8]. Prior to the secondary transition Neurog3 expression is quite low; however, at the start of the secondary transition its expression increases dramatically [31] and is subsequently lost quickly thereafter. This is precisely what we see in our data - low Neurog3 levels in the Pdx1 EGFP+ libraries, high expression in the Neurog3 EGFP+ libraries and diminishing expression in the TS22 and TS26 whole pancreas libraries, with no expression in the Neurog3 EGFP- or the adult islet or duct libraries. Neurod1, Isl1, Pax6 and Pax4 expression occurs subsequent to Neurog3, but unlike Neurog3 their expression is maintained in endocrine cell types [8]. In our data it is clear that the expression of all of these genes is most abundant in the Neurog3 EGFP+ libraries, or the islet library, as would be predicted. Ptf1a and Bhlhb8 (Mist1) are two transcription factors known to drive exocrine cell development. Ptf1a was found only in the TS22 whole pancreas library, and while low levels of Bhlhb8 were noted in the TS22 Neurog3 EGFP+ library, much higher levels were found in the duct cell library. Markers of mature exocrine cells showed peak expression in the TS26 whole pancreas or adult duct libraries, with moderate expression also in the islet library, suggesting a low level of exocrine cell contamination in this library. Glucagon expression peaked in the Neurog3 EGFP+ libraries, which is not surprising as Glucagon-positive cells are relatively abundant at these time points compared to in the adult islet. Iapp, Ins1 and Ins2 were all most abundant in the islet library, as was expected. The expression of these genes was also noted in the duct library, suggesting some level of islet cell contamination in this library. In sum, the expression profiles of these selected markers in our data match predictions based on their known expression profiles, indicating that our libraries accurately reflect the cell types and stages intended.

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