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

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Figure 1-1: Early embryo development from blastocyst stage to development through the primitive streak. A)Schematic representation of mouse embryonic development and signal gradients of BMP4, WNT, and nodal fromblastocyst to late-streak stages. The red line in the late streak-stage embryo marks a cross-section, which is shown inB). Names of marker genes expressed in a tissue-type are noted in brackets. B) Cross-section of embryo illustratinghow epiblast cells (in blue) move through the PS and form the definitive endoderm (DE) germ layer (in yellow)displacing the visceral endoderm (VE; in green). In between the DE and epiblast/ ectoderm, the mesoderm germ layer(in red) forms. Ant, anterior; AVE, anterior visceral endoderm; DE, definitive endoderm; Dist, distal; ExE, extraembryonicectoderm; ICM, inner cell mass; Post, posterior; Prox, proximal; PS, primitive streak; VE, visceralendoderm. Modified from (Gadue et al. 2005; Wolpert 2002).Cells start gastrulation by undergoing an epithelial-to-mesenchymal transition, enabling them tomove through the PS (Figure 1-1B, cross-section of the PS). FGF3, 4 and 8 are present in the PSand FGF4 and 8 are required for gastrulating cells to leave the PS (Sun et al. 1999). From chickenstudies it is suggested that FGF4 and 8 act as a chemo-attractant and a chemo-repellent,respectively on the migrating cells, driving cell movements after ingression through the PS(Wilkinson et al. 1988; Hebert et al. 1991; Niswander and Martin 1992; Sun et al. 1999; Yang etal. 2002b; Bottcher and Niehrs 2005). When cells have moved through the PS, they become eitherdefinitive endoderm (DE), displacing the visceral endoderm, or mesoderm forming anintermediate germ layer between the DE and the epiblast (Lawson et al. 1991; Tam et al. 1993;Carey et al. 1995). The remaining cells of the former epiblast become the ectoderm germ layer.Cells moving through the mid- and posterior PS early become different types of mesoderm,including some extra-embryonic mesoderm, influenced by BMP4 and low concentrations ofnodal (Parameswaran and Tam 1995; Kinder et al. 1999). The mesoderm expresses Fetal-likekinase 1 (Flk1) along with Noggin and Chordin. DE is formed from the anterior regions of the PS,influenced mainly by high concentrations of nodal. Here, Forkhead box A2 (Foxa2) is expressedduring gastrulation and is later found in the DE (Tam and Beddington 1992; Robb and Tam2004). A specific marker for the DE germ layer at this stage is the Sry-related HMG box gene 17(Sox17), which is not expressed in the other two germ layers, but is found also in the VE (Kanai-Azuma et al. 2002). The VE expresses the Sry-related HMG box gene 7 (Sox7), which is notfound in DE (Futaki et al. 2004; Seguin et al. 2008). DE cells migrating through the PS earlyduring gastrulation populate the anterior parts of the endoderm, the later foregut, and cells leavingthe PS late in gastrulation populate the posterior endoderm, the later hindgut (Tam andBeddington 1992).Gut tube patterning and regionalizationBy the end of gastrulation around E7.5 in the mouse, the embryo contains two major signallingcentres, the anterior VE (AVE) and the node. The AVE is found at the anterior side of the embryo(Figure 1-1A) and acts to restrict the PS structure and is involved in the head fold (Wells andMelton 2000). The node is found in the distal-most part of the embryo cylinder, the anterior limitof the PS, from where it directs DE patterning among other activities.The DE is patterned along the A-P axis through interactions with the anterior ectoderm, cardiacmesoderm, and notochord to form the anterior gut tube and with the node, lateral plate mesodermand PS to form the posterior gut tube (Wells and Melton 1999; Wells and Melton 2000). At E7.5anterior and posterior gut tube can be distinguished from one another not only by theirlocalization in the embryo, but also by their expression of marker genes. Mouse-Cerberus 1 (m-Cer1), Orthodenticle homeobox 2 (Otx2), Hematopoietically expressed homeobox (Hex) andHomeobox expressed in ES cells 1 (Hesx1) are expressed anteriorly and Cdx2 and certainmembers of the Hox family are expressed posteriorly. Dividing the forming gut tube into fourregions can illustrate how they re-localize to form the proper gut tube (Figure 1-2A&B; E8.5):Region I, the most anterior part, seems to fold over region II and form the ventral foregut, givingrise to the lungs, stomach, liver and ventral pancreas. Region II gives rise to the oesophagus,stomach, duodenum and dorsal pancreas. Similarly, region IV seems to fold over region III,giving rise to posterior trunk endoderm and hindgut, forming the large intestine. Region III givesrise to the midgut and trunk endoderm, forming the small intestine (Wells and Melton 1999).Closure of the gut tube happens as the anterior intestinal portal (AIP) and caudal intestinal portal8
(CIP) move rostrally (arrows in Figure 1-2A&B). Around E8.5 – E8.75 the embryo starts to turnso that the endoderm becomes situated on the inside, and the ectoderm on the outside of thedeveloping organism.Figure 1-2: Formation, folding and branching of the gut tube. A) The endoderm (yellow and light green) separatedfrom the mesoderm (red), ectoderm (blue) and PS (pink) in an E7.5 embryo. Roman numerals I–IV represent regionsof E7.5 endoderm that fate map to regions I–IV of the E8.5 gut in B). B) The forming gut tube (yellow) is seperatedfrom the remaining embryo. The foregut tube forms as region I folds over region II and migrates in a posteriordirection, whereas the hindgut tube forms when region IV folds over region III and migrates in an anterior direction(arrows in A). C) The formation of organ buds in a E10.5 embryo. D) Branching and maturation of the pancreas. A,anterior; AIP, anterior intestinal portal; CIP, caudal intestinal portal; D, dorsal; d. Panc, dorsal pancreatic bud; int,duodenum/intestine; Lu, lung; Li, liver; P, posterior; PS, primitive streak; St, stomach; V, ventral; v. Panc, ventralpancreatic bud. Modified from (Wells and Melton 1999).Instructive signals from the adjacent germ layers specify the A-P regions of the gut tube. Theanterior part of the gut tube has been shown to adopt a more posterior fate when exposed toposterior mesoderm or ectoderm in mouse embryos (Wells and Melton 2000). This has beensupported by studies in chicken, where anterior endoderm changes to a more posterior fate whentransplanted to a posterior site, but it cannot change to a more anterior fate when transplantedthere (Kumar and Melton 2003). These studies showed that increasing concentrations of FGF4and retinoic acid (RA) posteriorize the gut tube in mouse and chicken (Wells and Melton 2000;Dessimoz et al. 2006; Bayha et al. 2009). In the chick embryo BMP, activinA (a surrogate fornodal) and RA signalling could induce expression of the pancreatic foregut marker Pancreaticand duodenal homeobox 1 (Pdx1) in endoderm anterior to the pancreas (Kumar and Melton2003). Thus, signals originally implicated in the A-P specification of the neural tube were shownto have a similar function in the endoderm. The mouse gut tube can be specified by regionalmarkers, including Foxa2 expressed throughout the gut tube; Sox2 expressed in the thyroid,trachea and stomach regions; Pdx1 expressed in the duodenal and pancreatic regions; and Cdx2expressed in the posterior gut tube (as exemplified by the chicken cartoon in Figure 1-3; chickenCdxA = mouse Cdx2).9
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 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 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
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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
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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
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transplantation is the spread of an
Page 96:
AcknowledgementsThe work presented
Page 99 and 100:
Chambers, I., D. Colby, M. Robertso
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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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