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

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Figure 4-4: A high FGF4-concentration induces CDX2 + posterior gut tube. Flk1-LacZ cells differentiated for 5days in 100 ng/ml activin (Step 1) and 3 days in 25 ng/ml Wnt3a + 0, 10 or 100 ng/ml FGF4 (Step 2) were stainedfor FOXA2 and CDX2. 20× objective, scale bar indicates 50 µm.This resulted in numerous Sox2-LacZ + and SOX2 + cells as seen by β-Galactosidase stain andimmune-cytochemistry, respectively (Figures 4-2 and 4-3). Variation in the concentrations ofFGF4 and WNT3a did not seem to influence the numbers of Sox2-LacZ + or SOX2 + cells, aslong as both factors were present. We saw vast numbers of FOXA2 + cells throughout theconditions applied (Figure 4-3). FGF4 and WNT3a did not induce PDX1 + cells under anyconditions (Figure 4-3), but high concentrations of FGF4 in combination with WNT3a (5 or 25ng/ml) induced CDX2 + clusters in the culture (Figure 4-4). To be sure that we did not miss aweak expression of Pdx1 in the samples, we performed RT-PCR and saw only the housekeepinggene Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expressed (Figure 4-5).Thus, we concluded that FGF4 and WNT3a either alone or in combination were not enough todrive patterning of the DE into Pdx1-expressing posterior foregut cell types. We saw vastnumbers of Sox2-LacZ + and SOX2 + cells, indicative of an anterior DE-type. CDX2 wasinduced by 100 ng/ml FGF4, suggesting that this concentration is too high when aiming atPDX1-induction.52
Figure 4-5: Pdx1 cannot be detected by RT-PCR. RT-PCR for Pdx1 on E18,5 mouse pancreas and β-TC3, i.e.positive controls and Flk1-LacZ cells differentiated for 5 days in 100 ng/ml activin (Step 1) and 3 days in 25 ng/mlWNT3a ± 10 or 100 ng/ml FGF4 (Step 2). Gapdh is used as a house-keeping gene control. Loading in theindividual wells is described next to the agarose gel images.RA in combination with WNT3a and FGF4 induces posterior foregut cellsAs shown in paper I, figure 12, we could induce PDX1-expression by addition of 0.1 µM RAand intermediate levels of WNT3a (5 ng/ ml) and FGF4 (10 ng/ml) to Step 2 of thedifferentiation protocol. Under these conditions, we saw a vast number of FOXA2 + /SOX2 +cells and no CDX2-expressing cells. This indicates that RA, in combination with WNT3a andFGF4, induces cells of an anterior-intermediate gut fate expressing FOXA2 and SOX2 orPDX1, but no CDX2.Prolonged differentiation does not increase Pdx1-inductionTo be able to quantify the numbers of PDX1 + cells, we used a Pdx1-GFP cell line (Holland etal. 2006) and applied our 2-step protocol to this. We included KAAD-cyclopamine(cyclopamine; an inhibitor of SHH-signalling) in this protocol, as inhibition of SHH has provencrucial for Pdx1-induction in the developing gut tube (Hebrok et al. 1998).We generally saw induction of 2-4% Pdx1-GFP + cells when applying our standard protocol of0.1 µM RA, 5 ng/ml WNT3a and 10 ng/ml FGF4 (data not shown). This was not satisfactoryfor continued differentiation towards posterior foregut endoderm, and we speculated whetherour DE was still to immature to be patterned after 5 days of DE-induction. Thus, we introducedan extra step between endoderm induction and patterning: two days of culture with or withoutpatterning factors FGF10 and cyclopamine followed by combinations of RA, FGF10 andcyclopamine. In general, we saw a very low induction of Pdx1-GFP + cells in all conditions,ranging from 0,5-4% (Figure 4-6). There was a tendency for more Pdx1-GFP + cells with anincrease in factors applied, the combination of all three being the most potent. Also, the cellsgrown in FGF10 and cyclopamine in step 2 showed higher numbers of Pdx1-GFP + cells ingeneral. There seems to be little endogenous SHH-signalling in the DE cell population, asinhibition thereof did not improve our protocol. However, with a total amount of 2-4% Pdx1-GFP + cells, this protocol needs further optimization.53
Page 1:
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 54 and 55: Figure S6Figure S6: qRT-PCR analyse
Page 56 and 57: epithelium; Cdx2, expressed posteri
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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