FGF-signalling in the differentiation of mouse ES cells towards ...

4. Pxd1 inductionBackgroundThe work shown in Figure 12 of paper I was performed by Nina Engberg and me incollaboration. We worked together with other members of the lab on patterning the naïve DEinduced by high concentrations of activin. In this chapter, I will show additional data on thispatterning performed by myself, and refer to data performed by other members of the lab.The motivation behind this project departs from work by Mattias Hansson and Dorthe R.Olesen showing that they could differentiate mES cells to DE after 5 days of culture in 100ng/ml activin, referred to as ‘Step 1’ of the differentiation protocol. This DE was believed to beof a naïve type, as it did not stain positive for regional markers of the gut tube, such as SOX2,PDX1, NKX6.1 or CDX2 (Figure 4-1). We proposed that the DE could be patterned into cellsresembling posterior foregut endoderm, the region from which the pancreatic outgrowth occurs,when presented to the correct inducing signal.Figure 4-1: A high concentration of activin in Step 1 induces naïve DE. Flk1-LacZ cells were differentiated for 5days in 100 ng/ml activin and both differentiated cells mouse and tissue was stained for whole endoderm andregional markers: FOXA2, SOX2, PDX1, NKX6.1, CDX2 and the nuclear stain DAPI. Mattias Hansson,unpublished data.To pattern this naïve DE into posterior foregut endoderm, referred to as ‘Step 2’ of thedifferentiation protocol, we relied on data from mouse and chicken development. We chosespecific genes regionally expressed along the anterior-posterior (A-P) axis of the gut tube asmarkers of cell fate. These were Sox2, expressed anterior to the pancreatic region; Pdx1,expressed in the duodenum and pancreatic regions; Nkx6.1, expressed in the pancreatic49