Expression of Groucho/TLE proteins during pancreas development

<p>Abstract</p> <p>Background</p> <p>The full-length mammalian homologs of <it>groucho</it>, Tle1, 2, 3, and 4, act as transcriptional corepressors and are recruited by transcription factors containing an eh1 or WRPW/Y domain. Many transcription factors critical to pancreas development contain a Gro/TLE interaction domain and several have been shown to require Gro/TLE interactions for proper function during neuronal development. However, a detailed analysis of the expression patterns of the Gro/TLE proteins in pancreas development has not been performed. Moreover, little is known about the ability of Gro/TLE proteins to interact with transcription factors in the pancreas.</p> <p>Results</p> <p>We describe the expression of Gro/TLE family members, and of 34 different transcription factors that contain a Gro/TLE interaction motif, in the pancreas utilizing nine SAGE libraries created from the developing and adult pancreas, as well as the <it>GenePaint </it>database. Next, we show the dynamic expression of <it>Tle1</it>, <it>2</it>, <it>3</it>, <it>4, 5 </it>and <it>6 </it>during pancreas development by qRT-PCR. To further define the cell-type specificity of the expression of these proteins we use immunofluorescence to co-localize them with Pdx1 at embryonic day 12.5 (E12.5), Ngn3 at E14.5, Pdx1, Nkx2-2, Insulin, Glucagon, Pancreatic polypeptide and Somatostatin at E18.5, as well as Insulin and Glucagon in the adult. We then show that Tle2 can interact with Nkx2-2, Hes1, Arx, and Nkx6-1 which are all critical factors in pancreas development. Finally, we demonstrate that Tle2 modulates the repressive abilities of Arx in a β-cell line.</p> <p>Conclusion</p> <p>Although Tle1, 2, 3, and 4 show overlapping expression in pancreatic progenitors and in the adult islet, the expression of these factors is restricted to different cell types during endocrine cell maturation. Of note, Tle2 and Tle3 are co-expressed with Gro/TLE interaction domain containing transcription factors that are essential for endocrine pancreas development. We further demonstrate that Tle2 can interact with several of these factors and that Tle2 modulate Arx's repressive activity. Taken together our studies suggest that Gro/TLE proteins play a role in the repression of target genes during endocrine cell specification.</p

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

The expression profile of different developmental stages of the murine pancreas and predictions of transcription factor interactions, provides a framework for pancreas regulatory networks and development

Representative staining patterns for genes expressed in each of the identified expression profiles

Representative genes for each of the identified spatial expression profiles, including genes with known and previously un-described, or novel, staining profiles in pancreas development, are shown. For this, images of hybridization staining patterns for whole embryo sagittal sections were obtained from the website and magnified to show the pancreas (outlined in red). Relevant probe IDs can be found in Additional data file 4.<p><b>Copyright information:</b></p><p>Taken from "Identification of transcripts with enriched expression in the developing and adult pancreas"</p><p>http://genomebiology.com/2008/9/6/R99</p><p>Genome Biology 2008;9(6):R99-R99.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481431.</p><p></p

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 (); AAGTGGAAAAAAGAGGA (); TAGTTTTAACAGAAAAC (); ACCTTCACACCAAACAT (); AATGCAGAGGAGGACTC (); CAGGGTTTCTGAGCTTC (); TCATTTGACTTTTTTTT (); GATTTAAGAGTTTTATC (); CAGCAGGACGGACTCAG (); CAGTCCATCAACGACGC (); AGAAACAGCAGGGCCTG (); GACCACACTGTCAAACA (); CCCTGGGTTCAGGAGAT (); TTGCGCTTCCTGGTGTT (); ACCACCTGGTAACCGTA (); GCCGGGCCCTGGGGAAG (); CTAAGAATTGCTTTAAA (); GCCCTGTTGGTGCACTT (); TCCCGCCGTGAAGTGGA (). The libraries shown include: EGFP+ TS17 (P+ TS17); EGFP+ TS19 (P+ TS19); EGFP- TS20 (N- TS20); EGFP+ TS20 (N+ TS20); EGFP+ TS21 (N+ TS21); EGFP+ TS22 (N+ TS22); whole pancreas TS22 (WTS22); whole pancreas TS26 (WTS26); adult isolated ducts (Ducts); adult isolated islets (Islets).<p><b>Copyright information:</b></p><p>Taken from "Identification of transcripts with enriched expression in the developing and adult pancreas"</p><p>http://genomebiology.com/2008/9/6/R99</p><p>Genome Biology 2008;9(6):R99-R99.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481431.</p><p></p

Median plots of identified SAGE tag -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 -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: EGFP+ TS17 (P+ TS17); EGFP+ TS19 (P+ TS19); EGFP- TS20 (N- TS20); EGFP+ TS20 (N+ TS20); EGFP+ TS21 (N+ TS21); 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.<p><b>Copyright information:</b></p><p>Taken from "Identification of transcripts with enriched expression in the developing and adult pancreas"</p><p>http://genomebiology.com/2008/9/6/R99</p><p>Genome Biology 2008;9(6):R99-R99.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481431.</p><p></p

Specificity threshold accurately predicts spatial expression restriction

A plot of specificity (S) versus cumulative tag types represented shows the distribution of tags into tags with high (S > 0.1; top), medium (0.001 > S < 0.1, middle), and low (S < 0.001, bottom) S values. Representative hybridization staining patterns from TS22 whole embryo saggital sections obtained from are shown for each specificity group. Relevant probe IDs can be found in Additional data file 4. Arrows indicate the location of the pancreas (p).<p><b>Copyright information:</b></p><p>Taken from "Identification of transcripts with enriched expression in the developing and adult pancreas"</p><p>http://genomebiology.com/2008/9/6/R99</p><p>Genome Biology 2008;9(6):R99-R99.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481431.</p><p></p

SOTA clustering of temporal expression profiles from qRT-PCR analysis of 44 genes in pancreas development

qRT-PCR was used to determine the relative expression levels of the indicated genes during pancreas development at the TSs indicated. The relative level of expression of each gene was normalized and a SOTA analysis used to group the genes. Heatmaps of the relative expression levels of the genes in the SOTA groups, including the SOTA centroid, with peak expression in the islets, the TS26 developing pancreas, the TS21-TS26 developing pancreas, or the ducts are shown. The data shown are averages of the results obtained from pancreases from three separate litters (pancreases from an individual litter were pooled) or islet/duct collections with triplicate reactions from the separate RNA extractions.<p><b>Copyright information:</b></p><p>Taken from "Identification of transcripts with enriched expression in the developing and adult pancreas"</p><p>http://genomebiology.com/2008/9/6/R99</p><p>Genome Biology 2008;9(6):R99-R99.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481431.</p><p></p

Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression