MODULATION OF SONIC HEDGEHOG SIGNALING ALTERS CEREBELLAR DEVELOPMENT ...

187 Pages·2013·11.24 MB·English

by  Frances Cheng

MODULATION OF SONIC HEDGEHOG SIGNALING ALTERS CEREBELLAR
DEVELOPMENT AND MEDULLOBLASTOMA FORMATION

By

Frances Yun Cheng

Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
in
Cellular and Developmental Biology
December 2013
Nashville, Tennessee

Committee Members:
Professor Chin Chiang
Professor Christopher V.E. Wright
Professor Bruce D. Carter
Professor Robert J. Coffey
Professor Ethan Lee
Copyright © 2013 by Frances Yun Cheng
All Rights Reserved
To my parents and sister
To my husband and baby boy

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ACKNOWLEDGEMENTS
The work presented here was made possible by Public Health Service award
T32 GM07347 from the National Institute of General Medical Studies for the Vanderbilt
Medical-Scientist Training Program (MSTP), and the National Institute Of Neurological
Disorders And Stroke of the National Institutes of Health under Award Number
F31NS074638.
I would like to thank my advisor, Dr. Chin Chiang, who has always challenged,
focused, and supported me. His scientific perception, intuitive curiosity, and preciseness
have had a huge impact on my scientific training, and I will always feel grateful.
I would like to thank other faculty members who aided in my scientific growth
while at Vanderbilt University, particularly my committee chair, Dr. Chris Wright, who has
shown incredible support and care for my scientific training, and my committee
members: Dr. Robert Coffey, Dr. Bruce Carter and Dr. Ethan Lee. They have all
continually provided guidance and encouragement for my research progress and career
development.
The Vanderbilt MSTP program has been a steady and consistent source of
support and the strongest advocates for my success. I am thankful to Dr. Terry Dermody
for initially recruiting me into the program and for continual inspiration, as well as Dr.
Michelle Grundy, Dr. Jim Bills, Dr. Larry Swift and Melissa Krasnove for their continual
support. Thanks also to my MSTP mentor Dr. Pampee Young, who has been supportive
and provided me with personal and professional advice. The Vanderbilt Cell and
Developmental Biology department has been instrumental to my success in graduate
school and I’d like to thank the following members for their help: Elaine Caine, Kim
Kane, Dr. Anthony Tharp, Mark Wozniak, Ian McCullough.
Current and former members of the Chiang lab have been invaluable as friends
and colleagues, both in the lab and outside of it. Dr. Ying Litingtung has been full of
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scientific knowledge, ideas, and encouragement. My thanks particularly go out to
Jonathan Fleming, Kaitlyn Ryan, and Xi Huang (former member) for their support and
discussions on a daily basis both scientifically and personally, and who taught me many
of the things I know. This work would not be possible without them. I thank Jiang Liu,
Tatiana Ketova, and Annabelle Williams who were great colleagues. I thank former
mentors, Dr. Nenad Sestan, Dr. Mladen-Roko Rasin, and Dr. Vsevolod Gurevich who
guided and supported me.
I am certain I have the best MSTP class, whom I consider my family here in
Nashville, particularly Elizabeth, Eric, Tanner, John, Brian, Caroline, and Courtney.
Additionally, my friends from around the world have been there for me in the best and
worst of times and for their support and prayers I am eternally grateful: Emely, Victoria,
Jenny, Amy, Crissaris, Vanessa, Anna, Karen, Nana, Lynne, Connie, and many more.
I have been blessed with the most wonderful family. My husband, Johnny Lu, has
been by my side as a source of love and support from the moment I embarked on this
journey. I couldn’t have done any of this without him. Our greatest blessing, baby
Thomas, has given me so much joy and happiness. My dad (Ying), mom (Shirley), sister
(Diana) have always been an inspiration to me and my strongest supporters. I strive to
be more like them each day. Above all, I am thankful to my God for filling me with
strength, love, hope, and purpose (Isaiah 40:28-31).

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS………………………………………………………………………iv

LIST OF TABLES…………………………………………………………………………...…..vii

LIST OF FIGURES……………………………………………………………………….…… viii

CHAPTER

I. INTRODUCTION……..………………………...………………………………... 1

Cerebellar structure……………………………………………………. 1
Embryonic and early postnatal cerebellar development……..….…. 3
Sonic hedgehog signaling…………………………………….…….…. 12
Sonic hedgehog signaling in the cerebellum……………………..…. 15
Sonic hedgehog signaling and medulloblastoma……………….….. 17
Novel findings as elucidated by modulation of Shh signaling…..…. 23

II. WIDESPREAD CONTRIBUTION OF GDF7 LINEAGE TO CEREBELLAR CELL
TYPES AND IMPLICATIONS FOR HEDGEHOG-DRIVEN
MEDULLOBLASTOMA………………………...………………………………... 26

Introduction………..………………………………………..…..………. 26
Results………………………………………………………...…...……. 29
Discussion………………..………………………………….….………. 44
Materials and Methods……………………………………………...…. 52
Acknowledgements………………………………..……………….….. 54

III. GLIAL SONIC HEDGEHOG SIGNALING ACTIVITY IS REQUIRED FOR
PROPER CORTICAL EXPANSION AND CEREBELLAR
ARCHITECTURE………………………………………………………………… 55

Introduction………..……………………………………………………. 55
Results………………………………………………………..…...……. 58
Discussion………………..…………………………………….………. 88
Materials and Methods……………………………………………..…. 94
Acknowledgements……………………….…….………………….….. 96

IV. ANTAGONISM OF THE HEDGEHOG PATHWAY AT THE LEVEL OF GLI GENERAL DISCUSSION………...…………...………………………………... 84
TRANSCRIPTION BY SMALL MOLECULE 5-AMINOIMIDAZOLE-4-
CARBOXAMIDE-1-ß-4-RIBOFURANOSIDE (AICAR)………………………. 98

Introduction……..………………………………………………………. 98
Results…………………………………………………….…..…...……. 101
Discussion………………..………………………………….…….……. 122
Materials and Methods……………………………………………….... 127
Acknowledgements………………………………..……………….….. 130
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V. GENERAL DISCUSSION……………………...………………………………... 131

Summary…………..……………………………………………………. 131
Implications…………………………………………………..…...….…. 135
Future Directions………..………………………………….…….……. 141

REFERENCES…………………………………………………………………………….. 150

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LIST OF TABLES

Table Page

1. Medulloblastoma subgroups…………………………………………… 20

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LIST OF FIGURES

Figure. Page

1.1 Cerebellar structure……………………...……………….……………………... 3

1.2 Early cerebellar development…………………………………………………… 7

1.3 Shh signaling pathway…………………………………………………………... 12

2.1 Shh pathway activation in Gdf7-lineage cells leads to cerebellar
hyperplasia………………………………………………………………………... 30

2.2 Gdf7Cre/;SmoM2 mice develop medulloblastoma with CGNP features…….. 32

2.3 A subset of Gdf7-lineage cells express neural stem cell markers………….. 34

2.4 Roof plate cells give rise to a distinct population of cerebellar vermal radial
glia cells…………………………………………………………………………… 39

2.5 Gdf7-lineage cells in the cerebellar rhombic lip are Lmx1a+ neural
progenitor cells that give rise to CGNPs………………………………………. 42

2.6 Gdf7-lineage cells contribute to an extensive array of mature cerebellar
cell types………………………………………………………………………….. 44

2.7 In situ hybridization for Gdf7 expression………………………………………. 48

3.1 TNC-YFP-expressing cells are Bergmann glia. ……………………………… 61

3.2 TNCYFP-CreER;SmoF/- (SmoBG) mutants display a hypoplastic cerebellum. …. 63

3.3 Mutant EGL is largely agranular due to severely reduced CGNP
proliferation. ……………………………………………………………………… 64

3.4 Loss of Shh signaling is profound in CGNPs despite normal PC Shh
production…………………………………………………………………………. 67

3.5 Resident astroglial cells in the EGL do not contribute significantly to the
granular neuron population..……………………………………………………. 72

3.6 SmoBG mutants display altered BG arrangement and cytoarchitecture…….. 78

3.7 SmoBG mutants have disrupted alignment and dendritic arborization of
PCs………………………………………………………………………………… 81

3.8 SmoBG mutants exhibit aberrant Wnt signaling……………………………….. 86

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4.1 AICAR antagonizes Hedgehog (Hh) pathway activity in fibroblasts and
CGNPs…………………………………………………………………………….. 103

4.2 AICAR inhibits CGNP proliferation and growth of primary medulloblastoma
cells………………………………………………………………………………... 107

4.3 AICAR downregulates Shh signaling independently of the GSK3ß,
cholesterol, PKA, sirtuin, and mTOR pathways………………………………. 111

4.4 AICAR downregulates Shh signaling independently of the AMPK pathway. 112

4.5 AICAR acts downstream of Smoothened to inhibit Shh pathway activity….. 115

4.6 AICAR regulates Gli1 transcription…………………………………………….. 117

4.7 AICAR acts on human medulloblastoma cells to alter cell fate…...………… 120

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