Pancreatic tissue was isolated from FVF+ embryos at E 12.5 to E 15.5 in triplicates [1]. A single-cell suspension was obtained by trypsinization with 0.25% Trypsin and the specific incubated pancreatic tissue resuspension. Fluorescent-Activated cell sorting (FACs) was used to separate the FVF+ endodermal and FVF− non-endodermal cells. Total RNA was isolated using Trizol (Qiagen) and purified by using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. The Agilent 2100 Bioanalyzer had assessed RNA quality. The total RNA of three biological replicates for each embryonic stage was used to generate cRNA by two rounds of transcription. In general, a poly (dT) primer was utilized to synthesize cDNA from total RNA. In the next step, the cDNA amplified cRNA through T7 polymerase, using the T7 promoter sequence of the poly (dT) primer in the first round by random priming. Next, the cDNA was employed for the transcription into biotinylated cRNA, which hybridized to the 169 Affymetrix® array format according to the manufacturer protocol (GeneChip® Gene mouse 1.0 ST Array Card). Subsequently, processing of the Fluidics script FS450_0007 and scanning (GeneChip® Scanner 3000 7G Whole-Genome Association System) further lead to the probe set outcome.

The Affymetrix® Expression console normalized the robust multichip-analyses (RMA) on gene-level (ratio > 200 compared endodermal and non-endodermal). Comprehensive R based microarray analyses web frontend (CARMAweb) inducted gene-wise testing by Limma t-test and Benjamini-Hochberg multiple testing correction with a false discovery rate (FDR < 5%) offered statistically significant differential expression of 2921 probe sets [2]. Data were submitted to the Gene Expression Omnibus (GSE66856).

Subsequently, GO term enrichment, annotated through association of InterPro records with GO term, grouped 635 genes in the following clusters: extracellular/located at plasma membrane (304 genes), signaling molecules (222 genes), transcription factors (TF; 178 genes), cilium related (40 genes) and previously not described (42 genes). Afterwards, pancreatic-specific gene expression and localization were analyzed using the database GenePaint.org, respective gp3.mpg.de, which offers in silico in situs of whole mount embryosat E1 4.5. Pancreatic genes had been arranged according to their expression pattern in mesenchyme (non-epithelial), tip (exocrine), trunk (endocrine) and epithelium (pancreatic multipotent cells).

SNP of genes associated with T1D and T2D had been analyzed for their expression level during secondary transition and depicted in a heatmap (Figure 1). The heatmap represents the relative fold change in gene expression during the secondary transition stage between the non-endodermal and endodermal tissue compartments. The clustering of the genes was accomplished through extraction of gene symbols by Marullo et al. and Hakonarson et al. [3] [4]. Based on gene expression levels in the two tissue compartments we identified different clusters of the selected genes: transcriptionally regulated in both tissue populations or elevated either in the endodermal or the non-endodermal tissue compartment.

Heatmap shows clustering of 153 T1D- and T2D-associated genes according to their expression level during lineage commitment between E12.5 - 15.5. Predominantly, maturity-onset diabetes of the young (MODY) genes are upregulated in the endodermal compartment during pancreas organogenesis (Gck, Pdx1, NeuroD, Hnf1α, Hnf1ß). The black arrow marks the gene Ins2.

A subset of these T2D-associated genes which are regulated in our mRNA expression profile is illustrated in Table 1. Additional information of the mRNA localization (endodermal/non-endodermal), gene locus, chromosomal localization, the associated SNP and the disease as well as the related publications is provided as follows [4] - [12].

The GenePaint.org, respective gp3.mpg.dein silico in situ platform confirmed expression of mouse orthologues with associated SNPs in T2D shown in Table 1. Additional information about mRNA localization of the selected genes is obtained by inspection of the in situ whole mount embryos (see Figure 2).

Sagittal sections of whole mount embryos and magnification of the pancreatic region are shown. The genes associated with SNP in T2D as Kcnq1, Prox1, Hnf1ß, Gck, Foxa2 and Pdx1 exhibit epithelial in situ expression pattern.