Induction of the Isthmic Organizer and Specification of the Neural Plate Border Cédric Patthey Umeå Center for Molecular Medicine Umeå University, Umeå Sweden 2008 1 Front Cover: Photomontage showing how neural, neural crest and epidermal cells are already specified in the blastula stage chick embryo. Explants were taken as indicated in the ectoderm, cultured for 28 hr and 8µm sections were stained for Sox1 (red), HNK-1 (Green) or Cytokeratins (Blue). Copyright © Cédric Patthey ISBN: 978-91-7264-603-2 ISSN: 0346-6612 New Series No: 1197 Printed by Print & Media Umeå, Sweden, 2008 2 To Annika “Let us seek with the desire to find, and find with the desire to seek still more.” Saint Augustine 3 Table of contents Abstract......................................................................................................... 6 Papers in this thesis...................................................................................... 8 Abbreviations ............................................................................................... 9 Introduction................................................................................................ 10 Signaling factors and cell fate specification.............................................11 Why focus on the nervous system?..........................................................13 Description of the nervous system...........................................................13 Generation of cell diversity in the nervous system ..................................14 The Isthmic Organizer..............................................................................16 The neural plate border ............................................................................17 Specification of placodal and NC cells ....................................................19 Aims............................................................................................................. 21 Methodology ............................................................................................... 22 Results ......................................................................................................... 23 Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the Isthmic Organizer (Paper I)...........................................23 Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals (Paper II) .....................................26 Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate (Paper III).....................29 Discussion.................................................................................................... 35 A reasonable approach to study early vertebrate development................35 The IsO is induced by convergent Wnt and FGF signals ........................36 Source of IsO-Inducing signals................................................................38 Distinct time windows for IsO induction and maintenance.....................39 Mechanism of IsO establishment.............................................................39 Refinement of Wnt1 and Fgf8 expression domains .................................42 Context of neural plate border induction .................................................44 Temporal mechanism of Border induction ..............................................45 A novel patterning mechanism ................................................................47 The BMP gradient model.........................................................................48 Duration of BMP/Wnt signaling..............................................................49 Intracellular integration of BMP and Wnt signals ...................................50 4 Temporal Shift in response to BMP and Wnt signals..............................51 Border cells derive from neural cells .......................................................53 OLP cells derive from cells initially specified as NC cells......................53 Distinct roles of BMP and Wnt signals in NC cell specification.............55 Role of FGF signals in border induction..................................................56 Common origin of NC and placodal cells................................................57 Induction of other placodes......................................................................58 Are all NC cells induced simultaneously by the same mechanism?........59 Coordination of CNS and PNS development...........................................60 Conclusions ................................................................................................. 62 Acknowledgements..................................................................................... 63 References................................................................................................... 65 Supplementary Figures.............................................................................. 74 5 Abstract The vertebrate nervous system is extremely complex and contains a wide diversity of cell types. The formation of a functional nervous system requires the differential specification of progenitor cells at the right time and place. The generation of many different types of neurons along the rostro-caudal axis of the CNS begins with the initial specification of a few progenitor domains. This initial coarse pattern is refined by so-called secondary organizers arising at boundaries between these domains. The Isthmic Organizer (IsO) is a secondary organizer located at the boundary between the midbrain and the hindbrain. Although the function and maintenance of the IsO are well understood, the processes underlying its initial specification have remained elusive. In the present work we provide evidence that convergent Wnt and FGF signals initiate the specification of the IsO during late gastrulation as part of the neural caudalization process. The initial step in the generation of the nervous system is the division of the embryonic ectoderm into three cell populations: neural cells giving rise to the CNS, neural plate border cells giving rise to the peripheral nervous system, and epidermal cells giving rise to the outer layer of the skin. While the choice between neural and epidermal fate has been well studied, the mechanism by which neural plate border cells are generated is less well understood. At rostral levels of the neuraxis, the neural plate border gives rise to the olfactory and lens placodes, thickenings of the surface ectoderm from which sensory organs are derived. More caudally, the neural plate border generates neural crest cells, a transient population that migrates extensively and contributes to neurons and glia of the peripheral nervous system. How the early patterning of the central and peripheral nervous systems are coordinated has remained poorly understood. Here we show that the generation of neural plate border cells is initiated at the late blastula stage and involves two phases. During the first phase, neural plate border cells are exposed to Wnt signals in the absence of BMP signals. Simultaneous exposure to Wnt and BMP signals at this early stage leads to 6 epidermal induction. Wnt signals induce expression of Bmp4, thereby regulating the sequential exposure of cells to Wnt and BMP signals. During the second phase, at the late gastrula stage, BMP signals play an instructive role to specify neural plate border cells of either placodal or neural crest character depending on the status of Wnt signaling. At this stage, Wnt signals promote caudal character simultaneously in the neural plate border and in the neural ectoderm. Thus, the choice between epidermal and neural plate border specification is mediated by an interplay of Wnt and BMP signals that represents a novel mechanism involving temporal control of BMP activity by Wnt signals. Moreover, the early development of the central and peripheral nervous systems are coordinated by simultaneous caudalization by Wnt signals. 7 Papers in this thesis I. Olander S, Nordstrom U, Patthey C, Edlund T (2006). Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the Isthmic Organizer. Mechanisms of Development 123, 166-176. II. Patthey C, Gunhaga L, Edlund T (2008). Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals. PLoS ONE 3(2) e1625. III. Patthey C, Edlund T, Gunhaga L (2008). Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate. Submitted 8 Abbreviations B: Border BF-1: Brain factor 1 BMP: Bone morphogenetic protein C: Caudal CB: Caudal Border CNS: Central nervous system En1/2: Engrailed 1/2 FB: Forebrain FGF: Fibroblast growth factor HB: Hindbrain IsO: Isthmic Organizer Ker: Cytokeratins L: Lateral M: Medial MB: Midbrain mFrz8CRD: mouse Frizzled 8 cysteine rich domain MHB: Midbrain-hindbrain boundary OLP: Olfactory and lens placodes PNS: Peripheral nervous system R: Rostral RB: Rostral Border RT-PCR: Reverse transcription-polymerase chain reaction Sfrp: Soluble Frizzled-related protein Shh: Sonic hedgehog TGFβ: Transforming growth factor β Wnt: Wingless/Int 9 Introduction Introduction The human body is composed of approximately 1014 cells. Each cell is found at a correct position, exhibits appropriate structural and biochemical properties, and is coordinated within the whole organism. Particularly impressive is the nervous system which gives us the ability to sense and process