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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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Median plots of identified SAGE tag K-means cluster analysis using 14 clusters. We clustered 2, 536 SAGE tags with a count greater than 4 in one of the SAGE libraries and with a minimum specificity of 0.002 and that map unambiguously to a specific transcript or genome location into 14 clusters using K-means clustering using a PoissonC algorithm as described in the Materials and methods. The median normalized tag counts for the tags in each of the clusters is shown plotted against the indicated SAGE libraries. 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). A full list of the tags, the cluster they belong to, and their counts in each of the libraries is shown in Additional data file 2.
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Figure 3: Median plots of identified SAGE tag K-means cluster analysis using 14 clusters. We clustered 2, 536 SAGE tags with a count greater than 4 in one of the SAGE libraries and with a minimum specificity of 0.002 and that map unambiguously to a specific transcript or genome location into 14 clusters using K-means clustering using a PoissonC algorithm as described in the Materials and methods. The median normalized tag counts for the tags in each of the clusters is shown plotted against the indicated SAGE libraries. 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). A full list of the tags, the cluster they belong to, and their counts in each of the libraries is shown in Additional data file 2.

Mentions: We next wanted to group the tags based on their differential expression during pancreas development so as to segregate them based on their potential functional significance to the different stages and cell types represented by our libraries. First, a FOM analysis for the K-means algorithm with Euclidean distance was performed on normalized data, essentially as described [38]. Based on these results we performed a 14-cluster analysis using the PoissonC algorithm [39] with subsequent hand curation to finalize the clusters (Figure 3 and Additional data file 2).


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)

Median plots of identified SAGE tag K-means cluster analysis using 14 clusters. We clustered 2, 536 SAGE tags with a count greater than 4 in one of the SAGE libraries and with a minimum specificity of 0.002 and that map unambiguously to a specific transcript or genome location into 14 clusters using K-means clustering using a PoissonC algorithm as described in the Materials and methods. The median normalized tag counts for the tags in each of the clusters is shown plotted against the indicated SAGE libraries. 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). A full list of the tags, the cluster they belong to, and their counts in each of the libraries is shown in Additional data file 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Median plots of identified SAGE tag K-means cluster analysis using 14 clusters. We clustered 2, 536 SAGE tags with a count greater than 4 in one of the SAGE libraries and with a minimum specificity of 0.002 and that map unambiguously to a specific transcript or genome location into 14 clusters using K-means clustering using a PoissonC algorithm as described in the Materials and methods. The median normalized tag counts for the tags in each of the clusters is shown plotted against the indicated SAGE libraries. 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). A full list of the tags, the cluster they belong to, and their counts in each of the libraries is shown in Additional data file 2.
Mentions: We next wanted to group the tags based on their differential expression during pancreas development so as to segregate them based on their potential functional significance to the different stages and cell types represented by our libraries. First, a FOM analysis for the K-means algorithm with Euclidean distance was performed on normalized data, essentially as described [38]. Based on these results we performed a 14-cluster analysis using the PoissonC algorithm [39] with subsequent hand curation to finalize the clusters (Figure 3 and Additional data file 2).

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