The role of Arl13b in the maintenance of neural tube patterning and oligodendrocyte development Pubblico

Abstract

Both motor neurons and oligodendrocytes are essential for establishing neural
circuits in the central nervous system (CNS). They are distinct cell types, yet derived
from the same progenitor population, progenitors of motor neurons (pMN). In mouse,
pMN cells are specified at E8.5 in response to a low level of sonic hedgehog (Shh)
activity and differentiate as MNs at E9.5. Arl13b^hnn is a mouse mutant that disrupts a
ciliary small GTPase. Primary cilia are required for Shh signaling, and Arl13b^hnn disrupts
cilium architecture and Shh signaling. There is a constitutive low level of Shh activation
resulting in an expansion of pMN cells. The expansion persists to E12.5 leading
us to investigate whether Shh activity gradient is needed for the maintenance of neural
tube patterning over time, thus we induced a constitutive low level of Shh activity by
temporally deleting Arl13b. We defined a specific window when cells are sensitive to
changes in Shh activity. Surprisingly, cells that change their fate upon loss of Arl13b are
restored to normal over time suggesting that graded Shh response is not needed for
maintaining neural tube patterning. Our data suggest that Shh initially is an instructive
signal for specification, and then becomes a permissive signal for normal neural pattern.
At E12.5, pMN cells switch to become oligodendrocyte precursors (OLPs), and
platelet-derived growth factor receptor alpha (PDGFRa) signaling is important for OLP
specification. In Arl13bhnn, there are no OLPs at E13.5 before the embryos die. Primary
cilia are also implicated to be important for PDGFRa signaling, thus it is possible that
defective cilia result in impaired PDGFRa signaling in Arl13b^hnn. We deleted Arl13b at
E8.5 specifically in the pMN population, as well as at E10.5 in the CNS to target
progenitors before they become OLPs. We found that OLP specification is normal, but
oligodendrocyte differentiation and myelination are affected suggesting that Arl13b is
involved in the later steps of oligodendrocyte development. Taken together, our results
provide a better understanding of the maintenance of neural tube patterning, and of the
potential relationship between cilia and oligodendrocyte development.

Table of Contents

Table of Contents
Chapters

Chapter 1 Signaling, cilia, neural tube patterning, and oligodendrocyte development....1
1.1 Introduction ........................................................................................................2
1.2 The introduction of Arl13b protein and Arl13bhnn mouse mutant .................... 4
1.3 Neural tube patterning........................................................................................5
1.4 Shh signaling and cilia .......................................................................................9
1.5 Oligodendrocyte specification .........................................................................12
1.6 Signaling in oligodendrocyte development .....................................................14
1.7 Preview ............................................................................................................17

Chapter 2 Materials and methods ..................................................................................23
2.1 Generation of a conditional Arl13b allele ........................................................24
2.2 Genotyping for mouse strains ..........................................................................26
2.3 Preparation of genomic DNA from mouse tails ..............................................28
2.4 Southern blotting ..............................................................................................29
2.5 Immunofluorescence ........................................................................................30
2.6 Western blotting ...............................................................................................31
2.7 RNA in situ hybridization ................................................................................32
2.8 X-gal staining ...................................................................................................35
2.9 Tamoxifen injection .........................................................................................35

2.10 Quantitative analysis ......................................................................................35
2.11 Generation of mouse embryonic fibroblasts (MEFs).....................................36
2.12 Arl13b deletion in MEFs ...............................................................................37

Chapter 3 Shh acts first as an instructive morphogen and then as a permissive
signal in mammalian neural tube patterning ..........................................................43
3.1 Summary ..........................................................................................................44
3.2 Introduction ......................................................................................................45
3.3 Results ..............................................................................................................47
3.4 Discussion ........................................................................................................55

Chapter 4 The role of Arl13b in oligodendrocyte development ..................................83
4.1 Summary ..........................................................................................................84
4.2 Introduction ......................................................................................................84
4.3 Results ..............................................................................................................86
4.4 Discussion ........................................................................................................88
4.5 In the course of analysis of the Arl13b Olig1-Cre, we found minority of mutants
is identical to the null .......................................................................................91

Chapter 5 The prospective views of Arl13b in neural tube development and the
significance of Shh being a permissive role ...........................................................105
5.1 Summary of chapter 3 ....................................................................................106
5.2 The significance of Shh being an instructive and a permissive signal ..........106

5.3 Summary of chapter 4 ....................................................................................108
5.4 The potential roles of Arl13b in oligodendrocyte development ....................108
5.5 The potential role of Arl13b in neuroepithelial stem cells (NSCs) ...............110
5.6 Perspective and conclusion ............................................................................111

Bibliography ...................................................................................................................114

List of Figures

Figures

1.1 Cell signaling and neural tube patterning ..................................................18
1.2 Motile and primary cilia.............................................................................20
1.3 Cilia are required for Shh signaling ...........................................................21
1.4 Two models of how pMN cells become motor neurons and oligodendrocyte precursors (OLPs) ...22
2.1 Cloning sites of 5'arm, exon 2 of Arl13b, and 3' arm on pFlexible ..........38
2.2 The construct of a conditional Arl13b allele ..............................................39
2.3 The generation of a Arl13b^floxed allele ........................................................40
2.4 Exon two of Arl13b can be deleted upon Cre recombination ...................41
3.1 The generation of a conditional Arl13b allele............................................61
3.2 The rate of Arl13b protein turnover in vivo ...............................................63
3.3 Temporal deletion of Arl13b results in different neural tube patterning ...64
3.4 The kinetics of Arl13b protein deletion in conditional Arl13b knockout
MEFs is similar to in vivo ..........................................................................66
3.5 The localization of Smo in MEFs ..............................................................68
3.6 The pMN expansion is restored over time when Arl13b is deleted by
E9.25 and E9.5 .....................................................................................70
3.7 Rescue of patterning at E12.5 is not due to reactivation of Shh activity or
incomplete deletion of Arl13b ...................................................................72

3.8 The expression of the floor plate and p3 progenitors in different
conditional Arl13b knockout caudal neural tubes ......................................73
3.9 Abnormal Shh activity gradient results in distinct patterning in the
Arl13b^hnn rostral and caudal neural tube ....................................................75
3.10 Rostral neural tube displays normal patterning in all conditional Arl13b
knockout mutants ................................................................................77
3.11 Summary of phenotypic analyses ..............................................................79
3.12 The timeframe for the role of Shh in neural tube patterning .....................81
4.1 There are no OLPs in Arl13b^hnn at E13.5 ...................................................94
4.2 Oligodendrocyte specification and differentiation is normal when Arl13b
is deleted in the pMN lineage ....................................................................95
4.3 MBP and LINGO-1 expression is abnormal in Arl13b^Brn4-Cre .................96
4.4 An expansion of pMN cells and MNs in the minority of Arl13b Olig1-Cre ..98
4.5 Leaky Olig1-Cre expression ....................................................................100
4.6 Predictions of two models ........................................................................101
4.7 The expansion of pMN cells is resulted from Arl13b deletion regardless of
the number of Arl13b-lacking cells .........................................................102
4.8 Working model for the specification of distinct progenitors in the neural tube.......103
5.1 The potential role of Arl13b in neuroepithelial stem cells (NSCs) .........113

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Neuroscience
Parola chiave	Arl13b Primary cilia Neural tube patterning Sonic hedgehog
Committee Chair / Thesis Advisor	Caspary, Tamara, Emory University
Committee Members	Chen, Ping Helen, Emory University Wenner, Peter, Emory University Li, Xiao-Jiang, Emory University Chan, Anthony, Emory University

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