The role of cilia transport in oligodendrocyte development and regulation of PP2A activity Open Access
Umberger, Nicole Lynn (2014)
Published
Abstract
Platelet derived growth factor AA/αα (PDGF-AA/αα) signaling is essential for development of oligodendrocyte progenitors (OLPs) into mature oligodendrocytes (OLs), the cells which produce myelin to insulate axons of neurons in the central nervous system. OLPs are derived from the precursor motor neuron (pMN) domain in the neural tube, which later becomes the spinal cord, and specification of the pMN domain requires cilia. Cilia are microtubule based organelles, and are required for regulation of sonic hedgehog (Shh) signaling activity, which specifies the pMN domain. Arl13bhennin (Arl13bhnn) mouse embryos have short cilia, disrupted Shh activity, and, despite an expanded pMN domain, do not specify OLPs before embryos die during midgestation. Based upon this observation and previous connections of PDGF-AA/αα signaling to cilia, I asked if Arl13b and cilia are required for OLP development.
In this dissertation, I examine the role of Arl13b and of the cilia transport protein Ift88 in OLP development in vivo, and show that Ar13lb is not required for OLP specification or development, and that Ift88 is not required for postnatal OL development. To further dissect the role of Arl13b and cilia in PDGF-AA/αα signaling, I used an in vitro system and analyzed response to PDGF-AA stimulation in several cilia mutant cell lines. These experiments demonstrate that inhibited response to PDGF-AA stimulation in cilia mutant MEFs is due to up-regulation of mTORC1 signaling, and identify a novel role for PP2A in cilia signaling in vertebrates. Combined, my in vivo and in vitro results provide a more comprehensive understanding of the role of cilia in PDGF-AA/αα signaling.
Table of Contents
CHAPTER 1: Cilia, signaling, and oligodendrocyte development 1
1.1 Introduction 2
1.2 Cilia 2
Structure 3
Intraflagellar transport 4
1.3 IFT and cilia transport mutants 5
IFT172 5
IFT122 6
DYNC2H1 7
ARL13B 7
1.4 Sonic hedgehog 8
Signaling 8
Sonic hedgehog and cilia 10
Neural tube patterning 11
IFT, Shh, and neural tube patterning 12
1.5 Neural stem cell debate 14
Neural stem cell debate 14
1.6 PDGF-AA/αα signaling through PI3K/AKT and mTORC1 16
PDGFs and PDGFRs 16
Phosphatidylinositols and Phosphoinositide 3-kinase 17
AKT 18
mTORC1 19
PDGFAA/αα, and mTORC1 signaling in the context of cilia 21
PDGFAA/αα 21
mTORC1 21
1.7 Oligodendrocytes 23
Oligodendrocyte progenitor specification 23
Oligodendrocyte progenitor maturation 24
PDGF-AA/αα signaling in OLPs 25
Myelination 26
PDGF-AA/αα, cilia, and oligodendrocytes 26
1.8 Introduction to PP2A 27
Structure and regulation of PP2A 27
PI3K/AKT/mTOR signaling 28
Hh/Shh signaling 29
PP2A and cilia 30
1.9 Outstanding questions and preview 30
CHAPTER 2: MATERIALS AND METHODS 32
Brain tissue lysis 33
MEF lysis 33
Protein quantification 34
SDS PAGE and Western blotting analysis 35
Embryo dissections 36
Perfusion 37
Sectioning 37
Immunofluorescence 38
Mouse strains and genotyping 38
MEF preparation 41
PDGF-AA, LY294002, rapamycin, okadaic acid, and FTY720 treatments 42
PP2A activity assay 43
Chapter 3: The role of Arl13b and Ift88 in oligodendrocyte development in vivo 46
3.1 Summary 47
3.2 Introduction 47
3.3 Results 49
3.3.1 Generation of Arl13bΔOlig1-Cre mice, birth incidence, and post-natal weight 49
3.3.2 Deletion of Arl13b in Arl13bΔOlig1-Cre embryos 50
3.3.3 Arl13bΔOlig1-Cre embryos and pups fail to display phenotypes indicative of disrupted OL development 51
3.3.4 Repopulation of the pMN domain with Arl13b expressing Olig2+ progenitors (in Arl13bΔOlig1-Cre embryos at e12.5) 52
3.3.5 Generation and characterization of Ift88ΔOlig1-Cre mice 52
3.3.6 Rational for Nestin-Cre54
3.3.7 Generation of Arl13bΔNestin1-Cre mice and turnover of Arl13b in the neural tube 54
3.3.8 Arl13bΔNestin1-Cre mice do not display defects in prenatal OL development or early postnatal myelination 55
3.3.9 Arl13bΔNestin1-Cre mice do not display defects in postnatal myelination and develop cystic kidneys 56
3.3.10 Generation and characterization of Ift88ΔNestin1-Cre mice 56
3.4 Discussion 57
3.4.1 Benefits and disadvantages of Olig1-Cre and Nestin-Cre58
Table 3.1 58
3.4.2 Arl13b and Ift88 are not essential for OL development in vivo59
3.4.3 Support for a sequential progenitor model from Arl13bΔOlig1-Cre embryos 60
Chapter 4: Cilia transport regulates PDGF-AA/αα signaling via elevated mTOR SIGNALING AND DIMINISHED PP2A activity 62
4.1 Introduction 63
4.2 Results 65
4.2.1 PP2Ac localizes to the basal body of MEFs 65
4.2.2 P-AktT308 is Increased in Cilia Transport Mutant MEFs 66
4.2.3 PP2A Function is Disrupted in Cilia Transport Mutant MEFs 67
4.2.4 Total PP2A Activity is Similar in WT and Cilia Transport Mutant MEFs 69
4.2.5 mTORC1 Pathway Activity is Increased in Cilia Transport Mutant MEFs 69
4.2.6 Abnormal Response to PDGF Signaling in Cilia Transport Mutant MEFs 70
4.2.7 Rapamycin Treatment Restores PDGFRαα Levels and Response to PDGF-AA Stimulation 71
4.3 Discussion 73
Chapter 5: Perspectives77
5.1 Arl13b and Ift88 in oligodendrocyte development 78
5.2 PP2A and cilia trafficking 79
5.3 PDGF-AA/αα, Akt, and mTORC1 82
5.4 Final summary 84
FIGURES AND TABLES
Figure 1.2.1 Ciliary structure 85
Figure 1.2.2 IFT 86
Figure 1.4 Neural tube patterning 87
Figure 1.5 Mixed progenitor and sequential models of pMN domain specification 89
Figure 1.6 PDGF ligands and receptors 91
Figure 1.7.1 Stages of oligodendrocyte development 93
Figure 1.7.2 Myelination 95
Figure 1.8 PP2A 96
Figure 3.1 Arl13bΔOlig1-Cre observed vs expected, survival, postnatal weight 97
Figure 3.2 e10.5 neural tube of Arl13bΔOlig1-Cre embryos 100
Figure 3.3 MBP expression from p11 optic nerve of Arl13bΔOlig1-Cre pups 101
Figure 3.4 e16.5 neural tube of Arl13bΔOlig1-Cre embryos 102
Figure 3.5 e15.5 neural tube of Arl13bΔOlig1-Cre embryos 103
Figure 3.6 Ift88ΔOlig1-Cre observed vs expected 104
Figure 3.7 e12.5 neural tube of Ift88ΔOlig1-Cre embryos 106
Figure 3.8 Observed vs expected for Arl13bΔNestin-Cre and Ift88ΔNestin-Cre 107
Figure 3.9 e12.5 neural tube of Arl13bΔNestin-Cre embryos 108
Figure 3.10 e14.5 neural tube of Arl13bΔNestin-Cre embryos 109
Figure 3.11 e14.5 whole mount of Arl13bΔNestin-Cre embryos 110
Figure 3.12 PDGFRαα and Olig2 expression from p7 optic nerves of Arl13bΔNestin-Cre pups 111
Figure 3.13 e18.5 neural tube and Olig2, PDGFRα, and CNP expression from optic nerves of Ift88ΔNestin-Cre pups 112
Figure 4.1 PP2Ac localization and P-AktT308 levels in WT and cilia transport mutant MEFs 113
Figure 4.2 PP2A activity assay and un-methylated and methylated PP2Ac 115
Figure 4.3 mTORC1 signaling in WT and cilia transport mutant MEFs 116
Figure 4.4 Response to PDGF-AA stimulation with and without rapamycin 117
Figure S1 P-AktT308 localization in WT and cilia transport mutant MEFs 119
Figure S2 P-AktT308 levels of Dync2h1lln MEFs grown in 0.5% serum 120
Figure S3 Ift172wim MEFs do not form cilia during rapamycin treatment 122
Figure 5.3 PDGF-AA/αα, Akt, mTORC1, and PP2A interactions 123
REFERENCES 125
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