Post-Translational Modification of Pdx1 Figure 8. Pdx1 is phosphorylated in the developing chicken endoderm but S61 phosphorylation is not required for ectopic pancreas formation. Since the characteristic Pdx1 NIA profile was found early in mouse endoderm development (Fig. S3), we investigated the function of Pdx1 by in ovo electroporation of plasmids encoding Pdx1 and GFP into the endoderm of developing chicken embryos. One day following electroporation GFP expressing cells could be detected throughout the endoderm. A) OPT images showing the distribution of GFP expressing cells (green). Following dissociation, the GFP expressing cells were purified using FACS. Before sorting, the GFP expressing cells constituted 0.3% of the total number while the sorted fraction contained 53.5% GFP cells. B-D) The NIA profiles of the GFP positive fraction of embryos only electroporated with GFP (B), GFP and Pdx1 in the GFP negative fraction from embryos electroporated with GFP and Pdx1 (C) and the GFP positive fraction from embryos electroporated with both GFP and Pdx1 (D). In embryos electroporated with wild type Pdx1 the profile resembles the canonical Pdx1 profile. E-H) 3D image projections of confocal sections obtained from chicken embryos stained for Nkx6.1 (red), GFP (green) and Foxa2 (blue) by whole mount immunohistochemistry 72 hours after electroporation. In embryos electroporated with plasmids encoding GFP no ectopic Nkx6.1 expression could be detected (E). In embryos electroporated with wild type Pdx1 we observed the induction of ectopic pancreata and associated Nkx6.1 expression (F; arrowheads). Plasmids encoding Pdx1S61A (G) and Pdx1S61E (H) and also induced ectopic pancreata when electroporated into the endoderm. We also observed that the ectopic pancreata induced by wild type Pdx1 and S61A was associated with ectopic insulin expression (Fig. S4). dp (dorsal pancreas), st (stomach). Results are representative for two independent FACS analyses and at least three wholemount stainings for each Pdx1 construct. doi:10.1371/journal.pone.0035233.g008 being dissected and subjected to immunohistochemistry. The embryos were analyzed for the expression of GFP, Foxa2 (expressed in all endodermal cells) and Nkx6.1. Nkx6.1 is a marker of pancreas development and has previously been shown to be induced by Pdx1 [7,8]. In embryos electroporated with GFP alone the pancreas and anterior endoderm developed normally (Fig. 8E). Importantly, no expression of Nkx6.1 was detected in the endoderm outside the pancreatic buds. When Pdx1WT was electroporated we observed a strong induction of ectopic Nkx6.1 expression in the endoderm anterior to the pancreas (Fig. 8F). To investigate if the S61 phosphorylation has a role in pancreas development we looked for ectopic activation of Nkx6.1 expression PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35233