Figure 1-1: Early embryo development from blastocyst stage to development through <strong>the</strong> primitive streak. A)Schematic representation <strong>of</strong> <strong>mouse</strong> embryonic development and signal gradients <strong>of</strong> BMP4, WNT, and nodal fromblastocyst to late-streak stages. The red l<strong>in</strong>e <strong>in</strong> <strong>the</strong> late streak-stage embryo marks a cross-section, which is shown <strong>in</strong>B). Names <strong>of</strong> marker genes expressed <strong>in</strong> a tissue-type are noted <strong>in</strong> brackets. B) Cross-section <strong>of</strong> embryo illustrat<strong>in</strong>ghow epiblast <strong>cells</strong> (<strong>in</strong> blue) move through <strong>the</strong> PS and form <strong>the</strong> def<strong>in</strong>itive endoderm (DE) germ layer (<strong>in</strong> yellow)displac<strong>in</strong>g <strong>the</strong> visceral endoderm (VE; <strong>in</strong> green). In between <strong>the</strong> DE and epiblast/ ectoderm, <strong>the</strong> mesoderm germ layer(<strong>in</strong> red) forms. Ant, anterior; AVE, anterior visceral endoderm; DE, def<strong>in</strong>itive endoderm; Dist, distal; ExE, extraembryonicectoderm; ICM, <strong>in</strong>ner cell mass; Post, posterior; Prox, proximal; PS, primitive streak; VE, visceralendoderm. Modified from (Gadue et al. 2005; Wolpert 2002).Cells start gastrulation by undergo<strong>in</strong>g an epi<strong>the</strong>lial-to-mesenchymal transition, enabl<strong>in</strong>g <strong>the</strong>m tomove through <strong>the</strong> PS (Figure 1-1B, cross-section <strong>of</strong> <strong>the</strong> PS). <strong>FGF</strong>3, 4 and 8 are present <strong>in</strong> <strong>the</strong> PSand <strong>FGF</strong>4 and 8 are required for gastrulat<strong>in</strong>g <strong>cells</strong> to leave <strong>the</strong> PS (Sun et al. 1999). From chickenstudies it is suggested that <strong>FGF</strong>4 and 8 act as a chemo-attractant and a chemo-repellent,respectively on <strong>the</strong> migrat<strong>in</strong>g <strong>cells</strong>, driv<strong>in</strong>g cell movements after <strong>in</strong>gression through <strong>the</strong> PS(Wilk<strong>in</strong>son et al. 1988; Hebert et al. 1991; Niswander and Mart<strong>in</strong> 1992; Sun et al. 1999; Yang etal. 2002b; Bottcher and Niehrs 2005). When <strong>cells</strong> have moved through <strong>the</strong> PS, <strong>the</strong>y become ei<strong>the</strong>rdef<strong>in</strong>itive endoderm (DE), displac<strong>in</strong>g <strong>the</strong> visceral endoderm, or mesoderm form<strong>in</strong>g an<strong>in</strong>termediate germ layer between <strong>the</strong> DE and <strong>the</strong> epiblast (Lawson et al. 1991; Tam et al. 1993;Carey et al. 1995). The rema<strong>in</strong><strong>in</strong>g <strong>cells</strong> <strong>of</strong> <strong>the</strong> former epiblast become <strong>the</strong> ectoderm germ layer.Cells mov<strong>in</strong>g through <strong>the</strong> mid- and posterior PS early become different types <strong>of</strong> mesoderm,<strong>in</strong>clud<strong>in</strong>g some extra-embryonic mesoderm, <strong>in</strong>fluenced by BMP4 and low concentrations <strong>of</strong>nodal (Parameswaran and Tam 1995; K<strong>in</strong>der et al. 1999). The mesoderm expresses Fetal-likek<strong>in</strong>ase 1 (Flk1) along with Nogg<strong>in</strong> and Chord<strong>in</strong>. DE is formed from <strong>the</strong> anterior regions <strong>of</strong> <strong>the</strong> PS,<strong>in</strong>fluenced ma<strong>in</strong>ly by high concentrations <strong>of</strong> nodal. Here, Forkhead box A2 (Foxa2) is expresseddur<strong>in</strong>g gastrulation and is later found <strong>in</strong> <strong>the</strong> DE (Tam and Bedd<strong>in</strong>gton 1992; Robb and Tam2004). A specific marker for <strong>the</strong> DE germ layer at this stage is <strong>the</strong> Sry-related HMG box gene 17(Sox17), which is not expressed <strong>in</strong> <strong>the</strong> o<strong>the</strong>r two germ layers, but is found also <strong>in</strong> <strong>the</strong> VE (Kanai-Azuma et al. 2002). The VE expresses <strong>the</strong> Sry-related HMG box gene 7 (Sox7), which is notfound <strong>in</strong> DE (Futaki et al. 2004; Segu<strong>in</strong> et al. 2008). DE <strong>cells</strong> migrat<strong>in</strong>g through <strong>the</strong> PS earlydur<strong>in</strong>g gastrulation populate <strong>the</strong> anterior parts <strong>of</strong> <strong>the</strong> endoderm, <strong>the</strong> later foregut, and <strong>cells</strong> leav<strong>in</strong>g<strong>the</strong> PS late <strong>in</strong> gastrulation populate <strong>the</strong> posterior endoderm, <strong>the</strong> later h<strong>in</strong>dgut (Tam andBedd<strong>in</strong>gton 1992).Gut tube pattern<strong>in</strong>g and regionalizationBy <strong>the</strong> end <strong>of</strong> gastrulation around E7.5 <strong>in</strong> <strong>the</strong> <strong>mouse</strong>, <strong>the</strong> embryo conta<strong>in</strong>s two major <strong>signall<strong>in</strong>g</strong>centres, <strong>the</strong> anterior VE (AVE) and <strong>the</strong> node. The AVE is found at <strong>the</strong> anterior side <strong>of</strong> <strong>the</strong> embryo(Figure 1-1A) and acts to restrict <strong>the</strong> PS structure and is <strong>in</strong>volved <strong>in</strong> <strong>the</strong> head fold (Wells andMelton 2000). The node is found <strong>in</strong> <strong>the</strong> distal-most part <strong>of</strong> <strong>the</strong> embryo cyl<strong>in</strong>der, <strong>the</strong> anterior limit<strong>of</strong> <strong>the</strong> PS, from where it directs DE pattern<strong>in</strong>g among o<strong>the</strong>r activities.The DE is patterned along <strong>the</strong> A-P axis through <strong>in</strong>teractions with <strong>the</strong> anterior ectoderm, cardiacmesoderm, and notochord to form <strong>the</strong> anterior gut tube and with <strong>the</strong> node, lateral plate mesodermand PS to form <strong>the</strong> posterior gut tube (Wells and Melton 1999; Wells and Melton 2000). At E7.5anterior and posterior gut tube can be dist<strong>in</strong>guished from one ano<strong>the</strong>r not only by <strong>the</strong>irlocalization <strong>in</strong> <strong>the</strong> embryo, but also by <strong>the</strong>ir expression <strong>of</strong> marker genes. Mouse-Cerberus 1 (m-Cer1), Orthodenticle homeobox 2 (Otx2), Hematopoietically expressed homeobox (Hex) andHomeobox expressed <strong>in</strong> <strong>ES</strong> <strong>cells</strong> 1 (Hesx1) are expressed anteriorly and Cdx2 and certa<strong>in</strong>members <strong>of</strong> <strong>the</strong> Hox family are expressed posteriorly. Divid<strong>in</strong>g <strong>the</strong> form<strong>in</strong>g gut tube <strong>in</strong>to fourregions can illustrate how <strong>the</strong>y re-localize to form <strong>the</strong> proper gut tube (Figure 1-2A&B; E8.5):Region I, <strong>the</strong> most anterior part, seems to fold over region II and form <strong>the</strong> ventral foregut, giv<strong>in</strong>grise to <strong>the</strong> lungs, stomach, liver and ventral pancreas. Region II gives rise to <strong>the</strong> oesophagus,stomach, duodenum and dorsal pancreas. Similarly, region IV seems to fold over region III,giv<strong>in</strong>g rise to posterior trunk endoderm and h<strong>in</strong>dgut, form<strong>in</strong>g <strong>the</strong> large <strong>in</strong>test<strong>in</strong>e. Region III givesrise to <strong>the</strong> midgut and trunk endoderm, form<strong>in</strong>g <strong>the</strong> small <strong>in</strong>test<strong>in</strong>e (Wells and Melton 1999).Closure <strong>of</strong> <strong>the</strong> gut tube happens as <strong>the</strong> anterior <strong>in</strong>test<strong>in</strong>al portal (AIP) and caudal <strong>in</strong>test<strong>in</strong>al portal8
(CIP) move rostrally (arrows <strong>in</strong> Figure 1-2A&B). Around E8.5 – E8.75 <strong>the</strong> embryo starts to turnso that <strong>the</strong> endoderm becomes situated on <strong>the</strong> <strong>in</strong>side, and <strong>the</strong> ectoderm on <strong>the</strong> outside <strong>of</strong> <strong>the</strong>develop<strong>in</strong>g organism.Figure 1-2: Formation, fold<strong>in</strong>g and branch<strong>in</strong>g <strong>of</strong> <strong>the</strong> gut tube. A) The endoderm (yellow and light green) separatedfrom <strong>the</strong> mesoderm (red), ectoderm (blue) and PS (p<strong>in</strong>k) <strong>in</strong> an E7.5 embryo. Roman numerals I–IV represent regions<strong>of</strong> E7.5 endoderm that fate map to regions I–IV <strong>of</strong> <strong>the</strong> E8.5 gut <strong>in</strong> B). B) The form<strong>in</strong>g gut tube (yellow) is seperatedfrom <strong>the</strong> rema<strong>in</strong><strong>in</strong>g embryo. The foregut tube forms as region I folds over region II and migrates <strong>in</strong> a posteriordirection, whereas <strong>the</strong> h<strong>in</strong>dgut tube forms when region IV folds over region III and migrates <strong>in</strong> an anterior direction(arrows <strong>in</strong> A). C) The formation <strong>of</strong> organ buds <strong>in</strong> a E10.5 embryo. D) Branch<strong>in</strong>g and maturation <strong>of</strong> <strong>the</strong> pancreas. A,anterior; AIP, anterior <strong>in</strong>test<strong>in</strong>al portal; CIP, caudal <strong>in</strong>test<strong>in</strong>al portal; D, dorsal; d. Panc, dorsal pancreatic bud; <strong>in</strong>t,duodenum/<strong>in</strong>test<strong>in</strong>e; 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 <strong>the</strong> adjacent germ layers specify <strong>the</strong> A-P regions <strong>of</strong> <strong>the</strong> gut tube. Theanterior part <strong>of</strong> <strong>the</strong> gut tube has been shown to adopt a more posterior fate when exposed toposterior mesoderm or ectoderm <strong>in</strong> <strong>mouse</strong> embryos (Wells and Melton 2000). This has beensupported by studies <strong>in</strong> 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 transplanted<strong>the</strong>re (Kumar and Melton 2003). These studies showed that <strong>in</strong>creas<strong>in</strong>g concentrations <strong>of</strong> <strong>FGF</strong>4and ret<strong>in</strong>oic acid (RA) posteriorize <strong>the</strong> gut tube <strong>in</strong> <strong>mouse</strong> and chicken (Wells and Melton 2000;Dessimoz et al. 2006; Bayha et al. 2009). In <strong>the</strong> chick embryo BMP, activ<strong>in</strong>A (a surrogate fornodal) and RA <strong>signall<strong>in</strong>g</strong> could <strong>in</strong>duce expression <strong>of</strong> <strong>the</strong> pancreatic foregut marker Pancreaticand duodenal homeobox 1 (Pdx1) <strong>in</strong> endoderm anterior to <strong>the</strong> pancreas (Kumar and Melton2003). Thus, signals orig<strong>in</strong>ally implicated <strong>in</strong> <strong>the</strong> A-P specification <strong>of</strong> <strong>the</strong> neural tube were shownto have a similar function <strong>in</strong> <strong>the</strong> endoderm. The <strong>mouse</strong> gut tube can be specified by regionalmarkers, <strong>in</strong>clud<strong>in</strong>g Foxa2 expressed throughout <strong>the</strong> gut tube; Sox2 expressed <strong>in</strong> <strong>the</strong> thyroid,trachea and stomach regions; Pdx1 expressed <strong>in</strong> <strong>the</strong> duodenal and pancreatic regions; and Cdx2expressed <strong>in</strong> <strong>the</strong> posterior gut tube (as exemplified by <strong>the</strong> chicken cartoon <strong>in</strong> Figure 1-3; chickenCdxA = <strong>mouse</strong> 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 34 and 35: 290 M. Hansson et al. / Development
- Page 36 and 37: 292 M. Hansson et al. / Development
- Page 38 and 39: 294 M. Hansson et al. / Development
- Page 40 and 41: 296 M. Hansson et al. / Development
- Page 42 and 43: 298 M. Hansson et al. / Development
- Page 44 and 45: 300 M. Hansson et al. / Development
- Page 46 and 47: 302 M. Hansson et al. / Development
- Page 48 and 49: 304 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
- 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,