Apical Ectodermal Ridge (AER) activity and Limb outgrowth during Vertebrate Development Ana Raquel Viegas Tomás TESI DOCTORAL UPF 2010 Thesis Director: Joaquín Rodríguez León Centre de Medicina Regenerativa de Barcelona (CMRB) / Facultad de Medicina de la Universidade de Extremadura (UNEX) Aos meus Pais… AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT TABLE OF CONTENTS Acknowledgements ................................................................................................... ix List of Abreviations .................................................................................................. xi Summary ................................................................................................................. xiii Resumen.....................................................................................................................xv INTRODUCTION ......................................................................................................1 I Overview of vertebrate limb development..........................................................3 I.1 Proximodistal limb bud development...............................................................5 I.2 Limb bud positioning, initiation and early outgrowth......................................5 I.2.1 Establishment and maintenance of the fgf8/fgf10 positive feedback loop ....7 I.2.2 Retinoic acid and the promotion of Proximal-Distal (PD) outgrowth ..........9 I.3 Establishment of dorso-ventral (DV) polarity and patterning........................11 I.4 ZPA and the regulation of Anterior-Posterior (AP) patterning......................11 I.5 Axes coordination during limb development .................................................14 I.5.1 Proposed models for axes coordination during limb development .............15 II Developmental dynamics of the AER..............................................................17 III FGF signalling pathway regulation ................................................................20 III.1 Ras-MAPK/ERK pathway...........................................................................20 III.2 PI3 kinase/Akt pathway...............................................................................21 III.3 Modulators of FGF signalling .....................................................................22 III.3.1 Negative regulators: sef, sprouty and mkp3 ...............................................23 III.3.2 Positive regulators: flrt3.............................................................................24 IV The AER as a model for the study of epithelial renewal................................26 IV.1.1 The stemness marker, Oct4........................................................................28 AIMS..........................................................................................................................31 RESULTS ..................................................................................................................33 I Flrt3 as a key player in Limb Development......................................................37 Overview .................................................................................................................37 I.1 FLRT3 phylogenetic analysis.........................................................................39 I.2 Danio rerio’s flrt3 ..........................................................................................40 I.2.1 Gene expression pattern of flrt3 during zebrafish development .................42 I.2.2 Immunolocalization of FLRT3 during pectoral fin development ...............42 I.3 Gallus gallus’s flrt3........................................................................................43 I.3.1 Flrt3 expression is restricted to the AER and coincides with that of fgf8 and pERK activity..........................................................................................................43 I.3.2 Immunolocalization of FLRT3 during limb development ..........................46 I.3.3 Overexpression of flrt3 in the limb ectoderm induces ectopic ridges and enlargements of the pre-existent AER ....................................................................47 A.R.TOMÁS, 2010 v TABLE OF CONTENTS AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT I.3.4 Silencing of flrt3 affects the integrity of the AER ..................................... 52 I.3.5 Flrt3 expression is not regulated by FGF activity, although ectopic Wnt3a is able to induce flrt3.............................................................................................. 55 I.3.6 BMPs specifically inhibit flrt3 in the AER ................................................ 57 I.4 Gallus gallus’s flrt2....................................................................................... 58 II Oct4 sustains Apical Ectodermal Rigde renewal ............................................ 61 Overview ................................................................................................................ 61 II.1 Oct4 is expressed during limb development ................................................ 63 II.2 Cell dynamics in the AER............................................................................ 64 II.3 Oct4 overexpression expands the AER and enlarges the limb mesenchyme67 II.4 AER cell dynamics upon oct4 overexpression is altered ............................. 69 II.5 FGF signalling is needed to maintain oct4 expression................................. 71 II.6 The canonical WNT, WNT3A, controls oct4 expression in the AER ......... 74 II.7 BMPs negatively control oct4 expression prior to induction of cell death .. 74 II.8 RA activity controls oct4 expression but cannot induce an ectopic AER.... 75 II.9 Other candidate genes for oct4 co-regulation during AER renewal ............ 75 II.9.1 Retinoic acid metabolic enzymes, cyp26 and raldh2................................ 76 II.9.2 Sox14 ......................................................................................................... 77 II.9.3 p27Kip1........................................................................................................ 78 II.9.4 Lgr5 ........................................................................................................... 78 DISCUSSION ........................................................................................................... 81 I Flrt3 and vertebrate limb development ............................................................ 83 I.1 Flrt3 is essential for AER integrity and activity ........................................... 84 I.2 Flrt3 is necessary but not sufficient for proper AER formation and maintenance............................................................................................................ 85 I.3 Limb territorial borders are shiffted but identities are not altered upon Flrt3 overexpression in the limb ectoderm ..................................................................... 86 I.4 Flrt3 expression: the effect of FGF and WNT signalling ............................. 87 I.5 Flrt3 regulate AER’s BMP signalling through gremlin ................................ 88 I.6 Proposed model for flrt3 action in chick limb development ......................... 88 I.7 Flrt3 in zebrafish fin development ................................................................ 89 II Flrt3 in other FGF signalling centers.............................................................. 91 III Flrt2 ............................................................................................................... 93 IV AER cell dynamics ........................................................................................ 94 IV.1 Other epithelial cell renewal systems: the small intestine.......................... 95 V Oct4 in AER development and renewal.......................................................... 96 V.1 RA and BMPs negativelly regulate oct4...................................................... 97 V.2 Oct4 is upregulated through exogenous application of FGFs and WNT3A 99 VI Oct4 co-regulators of AER renewal ............................................................ 101 VI.1 Sox14 and p27, onsets for AER cell differentiation?................................ 101 VI.2 Lgr5 .......................................................................................................... 103 VII Proposed model for AER activity and renewal and involved players........ 105 CONCLUSIONS .................................................................................................... 107 A.R.TOMÁS, 2010 vi TABLE OF CONTENTS AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT MATERIALS AND METHODS...........................................................................111 I Embryo Models ...............................................................................................113 I.1 Staging, collection and processing of the biological material......................113 II Cloning of full-length and dsRNA constructs................................................114 II.1.1 Full-length flrt3........................................................................................114 II.1.2 Full-length oct4........................................................................................114 II.1.3 dsRNA against flrt3 .................................................................................115 III Chicken Embryo Manipulation.....................................................................116