We first assessed the expression of the Gro/TLE family members in nine Serial Analysis of Gene Expression (SAGE) libraries generated from the developing and adult pancreas 3132. This data revealed the differential expression of Tle1 through Tle6, with high levels of both Tle5 and Tle6, at various stages of pancreas development (Table 1). No tags unambiguously map to Tle4 so it could not be assessed. Since the Gro/TLE proteins interact with transcription factors containing an eh1 or WRPW/Y domain we assessed the expression of the 34 factors present in our pancreas SAGE libraries. Included in this group are several factors essential for proper endocrine cell specification and maturation such as Hes1, Foxa2, Nkx2-2, and Pou3f4 (Brn4) (Table 1). Arx and Nkx6-1, which are essential to proper α- and β-cell development respectively, and contain an eh1 domain, could not be detected as Arx does not produce an unambiguous tag while Nkx6-1 does not contain a NlaIII site and thus does not produce a SAGE tag.

We then used the GenePaint http://www.genepaint.org database to assess the staining patterns of these genes in the E14.5 pancreas. Tle 2 and 3 both showed modest staining throughout the epithelium and mesenchyme, while Tle1 staining was found predominantly in the mesenchyme (Figure 1A and Table 2). Expression of Tle4 could not be detected at this time point in the pancreas. Tle5 and 6 on the other hand showed strong staining throughout the pancreas epithelium. Informative stain was obtained for 22 of the 34 transcription factors containing an eh1 or WRPW/Y domain detected in our SAGE libraries and 11 of these showed stain in the pancreas (Figure 1B and Table 2). The remaining 11 factors for which informative staining was produced but no stain was seen in the pancreas typically showed counts only in the Pdx1 GFP+ libraries or the E12.5 Ngn3 GFP-, including Foxa1, Foxc1, Pax1, Pax3, and Tbx2, or in libraries representing later developmental stages, including Foxd3, Nkx2-5, Tbx20, and Tlx1. Data from the literature also provided evidence of the expression of an additional four of these factors, including Hes1 33, HlxB9 34, Nkx6-1 35, and Nkx6-2 36, which are not in the GenePaint database. Thus, in addition to our SAGE data, there is independent evidence demonstrating the expression of thirteen different eh1 or WRPW/Y transcription factors (Arx, Foxa2, Foxa3, Hes1, Hes6, Hhex, HlxB9, Nkx2-2, Nkx6-1, Nkx6-2, Pou3f4, Tcf1, and Tcf2), many of which have well established roles in endocrine pancreas development.

To determine the overall expression profiles of these genes in pancreas development we assessed their expression by qRT-PCR from E11.5 through to adult islets (Figure 2). The expression profiles of Tle1 and Tle2 were highly similar, and transcripts for both generally became more abundant as development progressed with peak expression in the adult islets. Tle3 expression dropped from E11.5 to E15.5 and was increased again at E18.5 and in adult islets. Tle4 expression also dropped at E13.5 and stayed low at the remaining time points analyzed, although levels in the adult islets were higher than those found at E13.5 to E18.5. Tle5 expression increased in abundance with development and highest levels were found in adult islets. Tle6 expression was almost inverted compared with the Tle3 and Tle4 profiles, with peak expression at E18.5 and relatively low levels in the islet and E11.5 samples.

To determine the cell type specificities of Tle1-4 we first analyzed their co-expression with Pdx1, whose expression marks the first appearance of cells capable of giving rise to the endocrine and exocrine pancreas as well as the duodenum 3738 (Figure 3 and Table 3). It was not possible to obtain Tle5 or Tle6 protein expression data since there are currently no suitable antibodies for these proteins. At this time point (E12.5) Tle1, 2, and 3 were expressed throughout the pancreatic epithelium and all Pdx1 positive cells were also positive for these three Gro/TLE proteins. A few Tle2 and Tle3 positive cells in the pancreatic epithelium were Pdx1 negative. These cells likely correspond to cells that have undergone early differentiation into glucagon positive cells. Tle4 was absent from the pancreatic epithelium and did not co-localize with Pdx1. All four of these Gro/TLE proteins were expressed in the mesenchyme surrounding the developing pancreas, with Tle1 and Tle3 expressed in a few such cells, while Tle2 and Tle4 were widely expressed.

During the secondary transition endocrine progenitors lose expression of Notch signaling components, such as Hes1, and begin to express the bHLH factor Ngn3. In neuronal development Tle1 expression is likewise lost during neural specification of progenitors. To see if a similar process occurs in the pancreas we co-localized Tle1-4 with Ngn3 in the E14.5 developing pancreas. Unlike at E12.5 where Tle1-3 were expressed throughout the pancreatic epithelium these factors clearly showed more restricted expression at E14.5 (Figure 4 and Table 4). At this time point Tle1 was absent from the pancreatic epithelium. Tle2 and Tle3 on the other hand both showed scattered expression through the epithelium. Also, both Tle2 and Tle3 co-localized with Ngn3 in a subset of Ngn3 positive cells. Interestingly, cells that were Ngn3 and Tle2 or Tle3 double positive, cells that were Ngn3 positive and Tle2 and Tle3 negative, and cells that were Ngn3 negative and Tle2 and Tle3 positive, were found (Figure 5). Tle4 was again absent from epithelial cells. Tle3 was only found in a restricted set of mesenchymal cells as observed at E12.5, while Tle1, 2, and 4 were widely expressed in the mesenchyme.

Beyond being implicated in controlling progenitor cell maintenance, Gro/TLE proteins also play a role in the maturation of a wide variety of specialized cell types. As previously mentioned many of the transcription factors that play significant roles in proper endocrine cell specification and maturation contain a Gro/TLE interaction domain. To determine which Gro/TLE family members are co-expressed with, and thus might interact with, these factors we co-localized the expression of Tle1 through 4 with Pdx1, Nkx2-2, Insulin, Glucagon, Pancreatic polypeptide, and Somatostatin, which between them mark the four predominant endocrine cell types in the pancreas, at E18.5.

Tle1 was found only in a few endocrine cells and its expression partially over-lapped only with Pdx1, which at this time point is restricted to β and δ cells. Tle1 expression was not seen in either Insulin or Glucagon positive cells. However it was found in cells that stained for the δ cell marker Somatostatin as well as in cells that stained for Pancreatic polypeptide (PP) (Figure 6). Tle2 was abundant in endocrine cells and all Pdx1 and Nkx2-2 positive cells were also Tle2 positive. Tle2 was also found in Insulin, Glucagon, Somatostatin and PP positive cells. Tle3 was nearly as broadly expressed as Tle2 and was found in all Pdx1 positive cells, and most Nkx2-2 positive cells. Tle3 was also found in Insulin, Glucagon, and Somatostatin positive cells but not in PP positive cells. Tle4 was found in rare endocrine cells, none of which expressed Pdx1 or Nkx2-2. Tle4 was also not found in Insulin, Glucagon, PP, or Somatostatin positive cells (Figure 6). A summary of these results can be found in Table 5.

Our qRT-PCR data suggests that the expression of Tle1, 2, and 3 peaks in the adult islets, while expression of Tle4 is almost as high as seen at E11.5. To determine if these genes show cell type specificity of expression within adult islets we next co-localized them with Insulin and Glucagon. Tle1, 2, 3 and 4 expression was quite broad within islets cell types, although Tle4 staining was noticeably weaker. Each of these proteins co-localized with Insulin in the majority of Insulin positive cells. However, in each case a number of cells positive only for Insulin were identified (Figure 7). All of the Glucagon positive cells were also Tle2 and Tle3 positive (Figure 7). The majority of Glucagon positive cells were also Tle1 and/or Tle4 positive. Thus clearly Tle1, 2, 3, and 4 are expressed in both of the major hormone producing cell types in the islet (Table 6). To characterize the Insulin or Glucagon positive Gro/TLE negative cells further we co-localized the Gro/TLE's using a pan-TLE antibody with PCNA, a marker of proliferating cells. All PCNA positive cells showed no or very weak Gro/TLE staining. Several Gro/TLE negative PCNA negative cells were also identified. We also assessed the co-localization of Tle1 through 4 with dolichos biflorus agglutinin (DBA) that marks ductal cells (data not shown). Only Tle2 and Tle3 were found in DBA positive cells. In acinar cells expression of all four of the Tle proteins was found, although Tle4 was present in only a very few cells.

Based on our expression profiling results Tle2 and Tle3 are the most likely candidates to be functional co-repressors for critical endocrine cell transcriptional repressors such as Nkx2-2, Nkx6-1, and others. In support of this, Tle3 interacts with Nkx2-2 in the pancreas 3 and knocking in a mutant Nkx2-2 containing the activation domain but not the Gro/TLE interaction domain produces a phenotype similar to the Nkx2-2 knock out 3 demonstrating that this domain is critical to proper Nkx2-2 function. However, the transcription factors that Tle2 interacts with in the developing pancreas are not known. Thus to determine if Tle2 interacts with transcription factors critical to key stages in pancreas development we used co-immunoprecipitation in the Min6 β-cell line. We first attempted to determine if Tle2, like Tle3, can interact with Nkx2-2 that is essential for the proper specification and maturation of α, β, and PP cells. Anti-Nkx2-2 blots of material immunoprecipitated using an anti-Tle2 antibody produced an ~30 kDa band that was not seen in controls (rabbit IgG used in immunoprecipitation) but was found in western blots of whole cell lysate (Figure 8A). Nkx6-1 has, like Nkx2-2, been shown to interact with Tle4 in in vitro binding assays 8. Nkx6-1 is critical for proper β-cell development and is known to act generally as a transcriptional repressor 5. We therefore wanted to determine if Tle2 could interact with Nkx6-1. Anti-Tle2 blots of material immunoprecipitated using an anti-Tle2 antibody produced an ~44 kDa band that was not seen in controls but was found in western blots of whole cell lysate (Figure 8B). Interestingly, the expected 46 kDa Nkx6-1 band could only be detected in the western blot 39. Next, we attempted to determine if Tle2 interacts with Hes1, a critical mediator of Notch signaling in pancreas development 40, which controls pancreas progenitor maintenance and specification 414243. Anti-Tle2 blots of material immunoprecipitated using an anti-Hes1 antibody produced an ~83 kDa band (Figure 8C). Recently Arx was shown to have the ability to convert both mature and developing β-cells into cells with an β-cell phenotype 44. Since it interacts with Tle1 45 we attempted to determine if it also interacts with Tle2. As Arx is not normally expressed in β-cells we co-transfected a 6xHis tagged Arx expression vector with a Flag tagged Tle2 expression vector. Anti-His blots of material immunoprecipitated using an anti-Tle2 antibody produced an ~63 kDa band that was not seen in controls but was found in western blots of whole cell lysate (Figure 8D). In sum these results indicate that Tle2 can interact with Nkx2-2, Nkx6-1, Hes1, and Arx.

Since Arx is able to convert mature β-cells into cells with an α-cell phenotype, we hypothesized that Gro/TLE-Arx interactions might be important to this ability. To assess this possibility we used a rat insulin promoter (RIP) reporter driving the expression of Enhanced Green Fluorescent Protein (EGFP). Transfection of the RIP-EGFP reporter with a Tle2 expression vector modestly reduced the green fluorescence intensity (Figure 9). However, transfection of the RIP-EGFP reporter with an Arx expression vector resulted in a 43% (p < 0.001) reduction in the mean green fluorescence intensity produced by the reporter as compared to transfection with the empty vector. Co-transfection of Arx with Tle2 resulted in a 61% (p < 0.001) reduction in the mean green fluorescent intensity produced by the reporter as compared to transfection with empty vector, a significant reduction from transfection with Arx alone (p < 0.01). Transfection with a Δeh1 Arx construct, which lacks the Gro/TLE interaction domain, resulted in a modest repression of the mean green fluorescence and co-transfection of this vector with the Tle2 vector did not alter the level of repression seen.

All mice were bred and maintained at the British Columbia Cancer Research Centre animal facility according to the guidelines of the Canadian Council on Animal Care. All protocols were approved by the University of British Columbia Animal Care Committee. Mice were housed in microisolator units, provided with Purina mouse food and autoclaved water ad libidum, and were maintained at 20°C ± 2°C under a light/dark cycle (light: 05:00–19:00 and dark: 19:00–05:00). Males were mated overnight with up to three females and females were checked for plugs before 9:30 the following morning. Plugged mice were considered to be 0.5 days post coitum (dpc). Islets were purified from 8–10 week old ICR males by collagenase digestion and gradient centrifugation as previously described 53.

SAGE data was analyzed using DiscoverySpace4 54. All SAGE libraries were generated and sequenced as part of the Mouse Atlas of Gene Expression 31 and Mammalian Organogenesis (MORGEN) projects. The data was filtered for sequence quality so that each tag had a 95% or greater probability of being correct, using the PHRED score quality assessment software 55. Tag to gene mapping was performed using the mouse Refseq, MGC, and Ensembl databases using the DiscoverySpace program. Tags were considered sense position matches if they mapped in the sense orientation to the gene and antisense matches if they mapped in the opposite orientation. A tag was considered unambiguous if it matched a single sense position gene in all of the databases, and ambiguous if it mapped to multiple genes in a sense position regardless of the mapping position. The specificity of tags was determined by first obtaining the counts for the tags in 205 different Mouse Atlas Libraries. From this the mean of the tag counts in all the libraries (M_a) was determined and compared to tag count in the library of interest (C_i) to obtain the mean ratio (M_r). The total number of libraries the tag was found in, or library count, was next determined (L_a) as was the total counts of the tag in the library under analysis (C_i). The specificity (S) was then calculated as: S = M_r log_1.3(C_i)/L_a. Thus tags with a high mean ratio that appear in relatively few libraries and are expressed more abundantly will have the highest specificities.

Probes for Tle1, 2, 3, and 4 as well as GAPDH were purchased from Applied Biosystems, Foster City, CA. An ABI 7500 real-time PCR system (Applied Biosystems) and Universal PCR Master Mix (Applied Biosystems) was used for all reactions. Six replicate cDNAs were obtained by reverse transcription (RT) of 1 μg of total RNA from newly isolated embryonic pancreas tissue or from Min6 cells for each RT. 10 ng of generated cDNA was used in each reaction with all reactions done in duplicate. Samples were normalized to Gapdh, and the fold increase compared to E11.5 pancreas or untreated Min6 cells (as appropriate) was calculated using 2^-ΔΔCt 56.

Images of in situ hybridization staining patterns for whole embryo sagittal sections were obtained from the GenePaint website (Visel et al., http://www.GenePaint.org). Higher magnification images of the area of the embryo containing the pancreas were obtained and the pancreas outlined. The brightness and contrast of some of the images was altered using Photoshop to better assess the staining pattern. Genes were then classified as showing trunk, tip, epithelial, mesenchymal or vasculature staining 57.

Immunohistochemistry was performed on E12.5, E14.5, E18.5 embryonic, or adult, pancreas cryo-sections sections using the following antibodies: 1/100 dilution of guinea pig anti-Insulin (Stem Cell Technologies Inc.); 1/500 dilution of guinea pig anti-Glucagon (Linco); 1/100 dilution of guinea pig anti-PP (Millipore); 1/1000 dilution of mouse anti-Somatostatin (Santa Cruz); 1/100 dilution of mouse anti-Nkx2-2 (Developmental Studies Hybridoma Bank); 1/100 dilution of mouse anti-Ngn3 (Developmental Studies Hybridoma Bank); 1/10,000 dilution of guinea pig anti-Pdx1 (kindly provided by Christopher Wright); 1/1000 dilution of rabbit anti-Tle1 (kindly provided by Stefano Stifani); 1/1000 dilution of rabbit anti-Tle2 (Santa Cruz); 1/1000 dilution of rabbit anti-Tle3 (Santa Cruz); 1/1000 dilution of rabbit anti-Tle4 (kindly provided by Stefano Stifani); 1/100 dilution of rabbit anti-pan TLE (Santa Cruz); 1/100 dilution of mouse anti-PCNA. Primary antibodies were detected using a 1/2000 dilution of Alexa 488 or Alexa 546 conjugated anti-rabbit, anti-mouse, anti-goat or anti-guinea pig (Invitrogen) antibodies as appropriate.

Min6 cells were maintained in high glucose (4,500 mg/l) Dulbecco's Modified Eagle's Medium (DMEM; StemCell Technologies Inc.) supplemented with 10% fetal calf serum and 5 mM L-glutamine. Immunoprecipitations were performed on 3 × 10⁶ Min6 cells or Min6 cells transfected with pCMS-6xHis-Arx and pCMS-Flag-Tle2, as appropriate. Cells were lysed and pre-cleared with 20 uL ProteinA-Sepharose (Rockland) to reduce background. Supernatants were incubated with 10 ug Rabbit Anti-Tle2 at 4° for 2 hours, and then with 20 uL of a 50% ProteinA-Sepharose slurry for 1 hour. Beads were washed three times in lysis buffer, resuspended in SDS sample buffer and electrophoresed on a 10% poly-acrylamide gel. Proteins pulled down using an anti-Tle2 (Santa Cruz) or anti-Hes1 (Santa Cruz) antibody were immunoblotted onto Immobilon-P membranes (Millipore) and detected with mouse anti-Nkx2-2 (Developmental Studies Hybridoma Bank), mouse anti-Nkx6-1 (Developmental Studies Hybridoma Bank), rabbit anti-Tle2 (Santa Cruz), or rabbit anti-His (Abcam). Negative controls to assess the specifity of the pulldowns included: rabbit IgG pulldowns and lysate only controls.

Assays were performed by transfecting 2 × 10⁵ Min6 cells with 600 ng of the appropriate vectors using Lipofectamine (Invitrogen) (pCMV-HA was used to adjust total DNA amounts) using the following amounts as appropriate: 300 ng of rat insulin promoter EGFP reporter plasmid, 50 ng of pCMS-Arx, 50 ng of pCMS-Arx Δeh1, 200 ng of pCMS-Flag-Tle2. 48 hrs after transfection the cells were harvested and single cell-suspensions prepared. Cells were then stained with 7-AAD (5 μg/ml) and analyzed by flow cytometry using a FACSCalibur flow cytometer and CELLQuest software (BD Pharmigen). Cells were gated based on the forward- versus side-scatter profile and on the 7-AAD versus forward scatter profiles to gate for viable cells, and a pCMS-HA only transfected sample was used to determine appropriate gating. The mean green fluorescence intensity was then determined for the samples using the CELLQuest software (BD Pharmingen).