Coordinating cell cycle exit and differentiation in the mammalian retina and its dependence on Rb By Marek Pacal A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Marek Pacal, 2011 Abstract Coordinating cell cycle exit and differentiation in the mammalian retina and its dependence on Rb Doctor of Philosophy (2011) Marek Pacal Department of Laboratory Medicine and Pathobiology University of Toronto Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either 1 side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin. 2 Acknowledgements I would like to thank to Dr. Rod Bremner for giving me the opportunity to learn in his lab, and Drs. Van Der Kooy and Huang for being on my student committee. I thank my parents for their support and patience and my dear Rachel for her love and understanding. Further, I am grateful for the generous financial support from the Vision Science Research Scholarship, Sandra and David Smith Graduate Student Award and Canadian Institute for Health Research. 3 Table of Contents Abstract ............................................................................................................................................ 1 Acknowledgements .......................................................................................................................... 3 Table of Contents ............................................................................................................................. 4 List of Figures and Tables ................................................................................................................ 7 List of Abbreviations ....................................................................................................................... 9 CHAPTER ONE: INTRODUCTION ............................................................................................ 10 1.1 Coordinating Division and Differentiation in Retinal Development ................................... 11 1.1.1 A few basics of cell cycle regulation .......................................................................... 13 1.1.1.1 Role of some core cell cycle components in retinal development .................... 16 1.1.1.2 Cyclins and Cdks in retinal development ......................................................... 17 1.1.1.3 The Rb family in retinal development .............................................................. 21 1.1.1.4 Ink4 CKIs and p19Arf in retinal development ................................................... 23 1.1.1.5 Cip/Kip CKIs in retinal development ............................................................... 24 1.1.1.6 E2fs in retinal development .............................................................................. 26 1.2 Separating Rate, Differentiation programs and Exit ............................................................ 27 1.2.1 Coupling INM to Cell Birth: “I need to get away” ..................................................... 28 1.2.2 Birth and Exit: “The Cog Model” vs. The Trigger theory” ........................................ 30 1.2.3 Birth and exit in frogs: “You walk, I’ll jump” ........................................................... 36 1.2.4 Coupling differentiation to cell cycle exit: “Let’s take the mystery tour” .................. 37 1.2.5 “We need a better map” .............................................................................................. 39 1.3 Cell Competition ................................................................................................................. 41 1.4 The role of Rb in differentiation .......................................................................................... 43 1.5 The Cell of Origin of Retinoblastoma ................................................................................. 45 CHAPTER TWO: TIMING OF CELL CYCLE MARKER SILENCING AND THE ONSET OF DIFFERENTIATION OF RETINAL GANGLION CELLS ......................................... 54 2.1 Introduction ......................................................................................................................... 55 2.2 Results ................................................................................................................................. 58 2.2.1 Ki67, but not Pcna or Mcm6, is confined to the NBL ................................................ 58 2.2.2 Ki67 labels all phases of the cell cycle in all RPCs .................................................... 59 2.2.3 A subset of Ki67 cells lack the pan-cell cycle markers Vsx2 and Ccnd1 ................... 60 2.2.4 Ki67 co-labels cells positive for presumed differentiation markers ........................... 62 2.2.5 Ccnd1 and Vsx1 extinction followed by induction of Isl1, Pou4f2, then Tubb3 in early RPCs .................................................................................................................. 63 2.2.6 The length of G2/M .................................................................................................... 65 2.2.7 Timing of Ki67 extinction in G0* cells ...................................................................... 66 2.2.8 Timing of expression of ganglion neuronal markers .................................................. 68 2.3 Discussion ........................................................................................................................... 70 2.3.1 Coordinating Exit and Differentiation: The Trigger Theory ...................................... 71 2.3.2 Evidence for RPCs biased towards the ganglion cell fate .......................................... 73 2.3.3 Induction and roles of Isl1 and Pou4f2 ....................................................................... 75 2.3.4 Ki67 remains in ganglion RTCs for a period of time after birth ................................ 76 4 CHAPTER THREE: TEMPORAL SEQUENCE OF EVENTS DURING ROD, AMACRINEAND BIPOLAR CELL BIRTHS IN THE MOUSE RETINA ....................... 94 3.1 Introduction ......................................................................................................................... 95 3.2 Results ................................................................................................................................. 97 3.2.1 Quantification of neuronal marker appearance with respect to cell birth ................... 97 3.2.1.1 Markers that label both RPCs and post-mitotic cells........................................ 98 3.2.1.2 Markers that label exclusively post-mitotic cells and can be used to detect rods and/or bipolar cells .................................................................................. 102 3.2.1.3 Markers that label exclusively post-mitotic cells and can be used to detect amacrine cells ................................................................................................. 110 3.3 Discussion ......................................................................................................................... 115 3.3.1 Panel of RTC markers .............................................................................................