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

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Figure S6Figure S6: qRT-PCR analyses of activin-stimulated ES cells after inhibition of Wnt signaling. Gsc Gfp/+ cells werecultured in 100 ng/ml activin for 5 days and (A) transfected with β-catenin siRNA as indicated or (B) treated withDkk1 as indicated. At the end of day 5 of the culture period RNA was prepared and qRT-PCR was performed tomeasure the relative abundance of Lhx1 and Chrd message.48
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 Mattias Hansson,unpublished data.To pattern this naïve DE into posterior foregut endoderm, referred to as ‘Step 2’ of thedifferentiation protocol, we relied on data from mouse and chicken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
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PhD thesisCand.scient. Janny Marie
Page 5: ResuméSukkersyge er en sygdom der
Page 9: Table of contents1
Page 12 and 13: ICMinner cell massIdInhibitor of di
Page 14 and 15: cell mass regenerates probably thro
Page 16 and 17: Figure 1-1: Early embryo developmen
Page 18 and 19: Figure 1-3: Regional expression of
Page 20 and 21: The pluripotent stateThe pluripoten
Page 25 and 26: There are four membrane-bound FGFRs
Page 27: 2. AimsThe aim of this study was to
Page 30 and 31: Developmental Biology 330 (2009) 28
Page 32 and 33: 288 M. Hansson et al. / Development
Page 50 and 51: Figure S2Figure S2: A subpopulation
Page 52 and 53: Figure S4Figure S4: Expression of T
Page 56 and 57: epithelium; Cdx2, expressed posteri
Page 58 and 59: Figure 4-4: A high FGF4-concentrati
Page 60 and 61: Figure 4-6: A 3-step protocol does
Page 63 and 64: 5. Paper IIFGFR(IIIc)-activation in
Page 65 and 66: AbstractProgress in embryonic stem
Page 67 and 68: variants in their Ig-like domain II
Page 69 and 70: Cell count and proliferation assayC
Page 71 and 72: influence on the numbers of Sox17-G
Page 73 and 74: undifferentiated cells, we found th
Page 75 and 76: FGF4, 5, FGF8b and FGFR1, are expre
Page 77 and 78: with EdU-stain (blue sample); and w
Page 79 and 80: Olsen, S.K., J.Y. Li, C. Bromleigh,
Page 81 and 82: FiguresFigure 1: Screen for FGFR-is
Page 83 and 84: Figure 3: Activation of FGFRb or FG
Page 87 and 88: Opposite, Figure 6: In the absence
Page 89 and 90: 6. General discussionEndoderm diffe
Page 91 and 92: Overall, the multitude of FGF-signa
Page 93 and 94: transplantation is the spread of an
Page 96: AcknowledgementsThe work presented
Page 99 and 100: Chambers, I., D. Colby, M. Robertso
Page 101 and 102: Hawkins, V.J. Wroblewski, D.S. Li,
Page 103 and 104: Nishikawa, S.I., S. Nishikawa, M. H
Page 105 and 106:
Tanimizu, N., H. Saito, K. Mostov,
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