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

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cell mass regenerates probably through an ability to ‘sense’ the total mass of insulin-producingcells and self-regulate accordingly (Hardikar 2004). Still, the success of these studies is low andsuch treatments will probably not be sufficient for curing diabetes.Replacement therapy by external sources of β-like cells holds the potential of an infinite supply ofdonor material for transplantation. The initial cell material may come from either somatic (oradult) stem cells or embryonic stem (ES) cells, or may in time come from patient-specific inducedpluripotent (iPS) cells. These cell types are described in detail in a later section.The immune system represents a major obstacle when discussing cell replacement-therapies.Patients would either have to be put on a life-long treatment of immune-suppressive drugs, withmany complications to follow, or other methods will have to be developed to overcome the attackof cell material by the recipient immune system (Rossini et al. 1999). Encapsulation of therapeuticcells is one such method. Despite limitations in nutrient supply to and transport of effectermoleculesfrom the transplanted cells, studies in rat show that they can convey normo-glycaemiain steptozotocin-induced diabetic rats (Omer et al. 2005). Another possibility is induction ofimmunological tolerance (mixed chimaerism), which has been shown possible by bone-marrowtransplantation in mice (Rossini et al. 1999; Blaha et al. 2005).Embryonic developmentTo appreciate the challenges of directed in vitro differentiation of ES cells, one must acquire athorough understanding of embryonic development. In this thesis, focus will be on mousedevelopment from zygote to pancreatic β cells, especially concentrating on the early development,as the work presented here is on mouse ES cell differentiation in the early steps towards β-likecells.From zygote to blastocystThe fertilized egg, the zygote, is a totipotent cell from which all tissues of the embryo proper, thegerm line and extra-embryonic tissues arise. In mice, the zygote moves into the uterine tract of thefemale and matures along the way. By embryonic day 3.5 (E3.5), it has developed into an ovalstructure, the blastocyst, consisting of an inner cell mass (ICM) at one end and the blastocoel atthe other end. The ICM gives rise to i) the epiblast (also called the primitive ectoderm), which willlater form the embryo proper and ii) facing the blastocoel, the primitive endoderm, which willgive rise to the visceral and parietal endoderm (PE), forming parts of the yolk sac and uterine walllining, respectively (Gardner 1983; Rossant 2004). Surrounding these structures is thetrophectoderm (TE), which will develop into the foetal parts of the placenta, the chorion. At E4.5the blastocyst implants into the uterine wall, gaining access to maternal blood vessels and therebya supply of nutrients and oxygen.The ICM expresses Octamer-4 (Oct4; encoded by Pou5f1), which is down-regulated in the TE(Nichols et al. 1998) and Nanog, which is maintained in the epiblast but down-regulated in the PE(Mitsui et al. 2003). Oct4 –/– and Nanog –/– embryos die due to a failure to form the ICM or theepiblast, respectively. Also, Sex determining region Y (SRY)-box 2 (Sox2) is present in the ICM,possibly as remnants of maternally deposited protein (Avilion et al. 2003). It is expressed by theembryo in the later epiblast and TE, where it, together with OCT4 and nanog inducestranscription of pluripotency-associated genes. The TE expresses Cdx2, a homeobox transcriptionfactor, which is required to form the placenta and to suppress transcription of Oct4 and Nanog(Chawengsaksophak et al. 2004; Strumpf et al. 2005). The PE expresses GATA-binding protein 6(GATA6; (Morrisey et al. 1998)). Recent studies have shown that cells in the ICM express bothnanog and GATA6 proteins prior to segregation into either epiblast or PE cells in a mosaic pattern(Chazaud et al. 2006). It is the expression level of fibroblast growth factor (FGF) that regulateseach cell’s fate in such that low levels of FGF generate epiblast cell formation and high levels ofFGF generate PE formation (Yamanaka et al. 2010). At this early stage, FGF4 and FGF receptor 1(FGFR1) are expressed in the ICM and knockout mice of both genes die prior to or at gastrulation(Deng et al. 1994; Yamaguchi et al. 1994; Feldman et al. 1995). Fgf4 expression is activated by6
SOX2 and OCT4. Later, the PE and epiblast transiently express Fgf5 (Haub and Goldfarb 1991;Hebert et al. 1991).Gastrulation and germ-layer formationAt E5.5 the epiblast is formed as a cup-like structure surrounded by the visceral endoderm (VE)which patterns the epiblast and initiates gastrulation (Figure 1-1A; (Rossant 2004)). It is at theposterior, proximal part of this cup that the primitive streak (PS) forms around E6.5 from where itstarts to migrate distally (Figure 1-1A). There is an anterior-posterior (A-P) patterning of theepiblast prior to gastrulation. Onset of PS formation is initiated by a gradient of the transforminggrowth factor β (TGFβ)-family member nodal and wingless-type MMTV integration site 3(WNT3) signalling from the posterior epiblast. Nodal generates a proximal-to-distal gradient andWNT3a forms a posterior-to-anterior gradient (Liu et al. 1999; Ben-Haim et al. 2006; Arnold andRobertson 2009). The nodal gradient moves distally, forming the PS along the way and finallyends at the distal-most part of the embryo, the node (Gadue et al. 2005). At the late streak stage,nodal expression is only found in the node where it forms a distal-to-proximal signal gradient. TheWNT3 expression pattern is restricted to the posterior epiblast during PS-formation. At the sametime, the TGFβ family member bone morphogenetic protein 4 (BMP4) from the extra-embryonicectoderm (ExE) signals to the adjacent epiblast. Thereby a proximal-to-distal signal gradientopposing that of nodal is formed (Lawson et al. 1999). The shape of these morphogeneticgradients is modulated by the reciprocal expression of antagonists or inhibitors, these being leftyand cerberus-like inhibiting nodal-signalling, dickkopf-related protein 1 (DKK1) inhibitingWNT3-signalling, and chordin and noggin inhibiting BMP4-signalling (Gadue et al. 2005).Thereby, the extension of the PS is restricted to the posterior side of the embryo.The primitive streak expresses the T-box transcription factor Brachyury (T) in migrating cells ofthe PS and Mix1 homeobox-like (Mixl1), along with Even-skipped homeobox homolog 1 (Evx1)(Figure 1-1A; (Bastian and Gruss 1990; Dush and Martin 1992; Kispert and Herrmann 1994; Nget al. 2005)). Goosecoid (Gsc) is expressed in the progressing PS and localises to the anteriorstreak (Blum et al. 1992).7
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 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 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
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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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