The role of Arl13b in the maintenance of neural tube patterning and oligodendrocyte development 公开
Su, Chen-Ying (2011)
Published
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. Arl13bhnn is a mouse mutant that disrupts
a
ciliary small GTPase. Primary cilia are required for Shh signaling,
and Arl13bhnn
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
Arl13bhnn. 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 Arl13bfloxed 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
Arl13bhnn 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 Arl13bhnn 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
Arl13bBrn4-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
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