Embryonic Stem Cells Assume a Primitive Neural Stem Cell Fate in the Absence of Extrinsic Influencesfor the degree of Doctor of Philosophy
Graduate Department of Medical BioPhysics
University of Toronto
University of Toronto 2009
Abstract
In this thesis, I describe studies identifying and characterizing two putative stem cell
populations of the neural and pancreatic lineages. The mechanisms governing the
emergence of the earliest mammalian neural cells during development and the ontogeny
of neural stem cells remain incompletely characterized. A default mechanism has been
suggested to underlie neural fate acquisition, however an instructive process has also
been proposed. I utilized mouse ES cells to explore the fundamental issue of how an
uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic
signals, and what cellular fate will result. Individual ES cells were found to rapidly
transition directly into neural cells by a default mechanism, a process shown to be
independent of suggested instructive factors. Further, I provide evidence that the default
neural identity is that of a primitive neural stem cell, the earliest identified stem cell of
the neural lineage. The exiguous conditions used to reveal the default state were found to
present primitive neural stem cells with a survival challenge, which could be mitigated by
survival factors or genetic interference with apoptosis. I also report the clonal
identification of multipotent precursor cells, PMPs, from the adult mouse and human
pancreas. These cells proliferate in vitro to form clonal colonies and display both
pancreatic and neural cell multipotentiality. Importantly, the newly generated cells
demonstrate glucose-dependent Ca2+ responsiveness and regulated insulin release. PMP
colonies do not express markers of embryonic stem cells, nor genes suggestive of
mesodermal or neural crest origins. Moreover, genetic lineage-labeling experiments
excluded the neural crest, and established the embryonic pancreatic lineage, as the
developmental source of PMPs. The PMP cell was further found to express insulin in
vivo, and insulin+ stem cells were shown to contribute to multiple pancreatic and neural
cell populations in vivo. These findings demonstrate that the adult mammalian pancreas
contains a population of insulin+ multipotent stem cells, capable of contributing to the
pancreatic and neural lineages. In the final section of this thesis, I consider the
relationships between neural and pancreatic tissues, as well as discussing the relevance of
these two novel stem cell populations.
Acknowledgments
No man is an island, and no man’s accomplishments are achieved in a vacuum*. There
are many people who have contributed in various ways to effect the successful
completion of this thesis. Foremost, I would like to thank my supervisor, Derek, for
providing just the right yolk to enable the flourishing and development of the egg that is
my research work. Derek is genuinely enthusiastic about science, with an energetic yet
easygoing supervisory approach that provided superb guidance while allowing me to
develop my own research skills. Perhaps most importantly, he maintains a healthy sense
of humour, which, I’m sure, allowed him to put up with me over the years. Thank you.
I would also like to thank all the members of the van der Kooy lab. It has truly been a fun
and productive experience working with people who know how to both work hard and
play hard. It has been a pleasure to be part of this wonderful team. I would like to
specifically acknowledge all of the support provided by our lab technicians, Brenda
Coles-Takabe and Sue Runciman, without whom the lab would surely fall apart.
I would like to express my gratitude to my generous colleagues and collaborators who
have provided conceptual insights, technical advice, reagents, and animal strains.
Last, but certainly not least, I would like to thank my family and friends for their love and
encouragement, which I certainly reciprocate. I am sure that I would be even less sane
without your support.
iii
List of Abbreviations x
Chapter I: General Introduction 1 Development of the Early Embryo and Embryonic Stem Cells 4
Neurulation and Neural Induction 18
Brain Development and Neural Stem Cells 25
Pancreatic Development, Regeneration, and Adult Pancreatic Precursor Cells 30
Chapter II: Primitive Neural Stem Cells: The Default Fate of Embryonic Stem Cells 39
Summary
Introduction
Results
Discussion
Chapter III: Identification of Multipotent Precursors From the Adult Mouse Pancreas 89
Summary
Introduction
Results
Discussion
iv
Chapter IV: The Adult Mouse and Human Pancreas Contain Rare Multipotent Stem Cells that Express Insulin 141
Summary
Introduction
Results
Discussion
Neural-Pancreatic Relationship 198
Are Primitive NSCs and PMPs Bona Fide Stem Cells? 200
Are Primitive NSCs and PMPs Developmentally, Physiologically,
or Therapeutically Relevant? 206
Chapter I
36
Chapter II
Figure 2.1 ES Cells Rapidly Transition into Neural Cells When Placed in Minimal Conditions
48
Figure 2.2 The ES Cell Neural Transition Occurs by Default Without Requirement for Instructive Factors
51
Figure 2.3 Primitive Neural Stem Cells Emerge from the ES Cell Default Neural Pathway
54
Figure 2.4 Expanded RT-PCR Analysis of Primitive Neurospheres Confirms Their Neural Precursor Character
57
Figure 2.5 Primitive Neural Stem Cell Proliferation is Dependent on Autogenously-Produced FGF Signalling
61
Figure 2.6 A Survival Challenge Due to the Minimal Culture Conditions Limits the Number of ES-Derived Neural Precursors that Survive to Form Primitive Neurospheres
65
70
74
Figure 2.9 ES Cell-Based Model Describing the Ontogeny of Neural Stem Cells
80
vi
Chapter III
Figure 3.1 PMP Colonies are Formed from Progenitors Present in Adult Pancreatic Islet and Duct Cell Isolates, and Express Markers Characteristic of Both Neural and Pancreatic Precursors
95
Figure 3.2 PMP Colonies Generate All Three Major Neural Cell Lineages
100
Table 3.1
Comparison of the mean percentages of neural and pancreatic cell progeny generated from murine adult pancreas (PMP) colonies and adult forebrain-derived neurospheres.
101
Figure 3.3 Progeny from Two Distinct Embryonic Primary Germ Layers Are Generated By Single, Clonally-Derived PMPs that are Present in Islet and Ductal Cell Isolates
104
Figure 3.4 Differentiated PMP Colonies Contain Cells that Co- Express Pax6 and C-peptide
107
Figure 3.5 Insulin+ Cells Generated De Novo From PMPs Demonstrate Glucose-Stimulated Ca2+ Responses and Glucose-Stimulated Insulin Release
110
Figure 3.6 PMP Colonies Generate Multiple Islet Endocrine Subtypes and Exocrine Cells
113
Figure 3.7 PMPs Are Not General Endodermal or Mesodermal Precursors, nor Are They ES-Like Stem Cells or Neural Crest Precursors
116
Figure 3.8 PMPs Are Present in Both Nestin+ and Nestin- Cell Fractions from both Islet and Ductal Cell Isolates
120
Comparison of the gene expression profile of mouse PMP colonies and brain-derived neurospheres by RT-PCR analysis, for both undifferentiated and differentiated conditions.
124
vii
Chapter IV
Figure 4.1 Neural Crest-Lineage Cells do not Form PMP Spheres 148
Figure 4.2 Transgenic MIP-GFP Mice Exhibit Flourescence in Insulin- Expressing Cells
151
Figure 4.3 FACS Analysis Showed that the Sphere-Initiating PMP Cell Population was Contained within the Insulin+/Glut-2-low Fraction
154
Figure 4.4 Insulin-Expressing Cells from MIP-GFP Mice Proliferate to Form PMP Spheres which are Multipotent in the Pancreatic and Neural Lineages
156
Figure 4.5 PMP Spheres were Generated by Cells in the GFP-Positive and GFP-Negative Cell Fractions from RIP-Cre-ER x Z/EG Mice
159
Figure 4.6 Spheres from a Single GFP+ Cell from RIP-Cre-ER x Z/EG Mice are Multipotential in the Pancreatic and Neural Lineages
161
Figure 4.7 Insulin-Positive Cells Generate Multiple Pancreatic and Neural Cell Types in Vivo
164
Figure 4.8 In Vitro Tamoxifen-Induction Initiates GFP Expression Exclusively in Insulin-Expressing Cells, which can then Generate Multiple Cell Types
167
Figure 4.9
Human Islet Tissue Contains Cells Capable of Proliferation to Form Self-Renewing Sphere Colonies, which Generate Multiple Pancreatic and Neural Cell Types upon Differentiation
170
Table 4.1 Gene Expression in Undifferentiated Human PMP Spheres. 171
Figure 4.10 Differentiation of Human PMP Spheres Yielded Somatostatin-Expressing Cells with a Neuronal Morphology
174
viii
Table 4.2 Gene Expression in Differentiated Human PMP Spheres. 175
Figure 4.11 Undifferentiated Human PMP Sphere Cells Do Not Display ES Cell Markers
177
Figure 4.12 Cells Generated de novo from Human Spheres Demonstrate Regulated Glucose-Stimulated Insulin Release
180
Figure 4.13 Expression of Ngn-3 in Insulin-Positive Cells of Wild-Type Normal Mice
185
ix
x
AIF apoptosis-inducing factor AC adenylate cyclase Apaf1 apoptotic protease activating factor-1 AVE anterior visceral endoderm BMP bone morphogenic protein cAMP cyclic adenosine monophosphate cGMP cyclic guanosine monophosphate DG dentate gyrus E embryonic day EB embryoid body EGF epidermal growth factor EMT epithelial-to-mesenchymal transition ES embryonic stem FACS fluorescence-activated cell sorting FBS fetal bovine serum FGF fibroblast growth factor GC guanylate cyclase ICM inner cell mass LIF leukemia inhibitory factor MAPK mitogen-associated protein kinase NAC N-acetyl-L-cysteine NC neural crest NS neurosphere NSC neural stem cell PBS phosphate buffered saline pCPT 8-(4-chlorophenylthio) PE primitive endoderm PMP pancreas-derived multipotent precursor RT-PCR reverse transcriptase - polymerase chain reaction SFM serum-free media SVZ subventricular zone TE trophectoderm TGF transforming growth factor beta
CHAPTER I
General Introduction
-Ralph Waldo Emerson
Stem cells are “founder” cells that function to give rise to the multitude of various
differentiated cells during development, as well as in the maintenance of adult tissues
during normal cell turnover, repair, and regeneration. Current definitions of stem cells
ascribe to them the cardinal properties of self-renewal, i.e. maintenance of the stem cell
identity in at least one of the two daughter cells after a division event, and
multipotentiality, i.e. the capacity to yield multiple mature differentiated cell types. Stem
cell potency may be very broad, as with pluripotent stem cells derived from the early
embryo, or relatively restricted, as with adult tissue-specific stem cells. Within this thesis,
I use the term “stem cell” to describe cells demonstrated to possess the hallmark stem cell
properties described above, and the term “progenitor” to describe cells possessing a
limited version of these properties, often with limited potency or having low self-renewal
ability. The term “precursor” is used as a general term encompassing both of these.
In this thesis, I describe the discovery of two different putative stem cell
populations present at opposite ends of the developmental spectrum, i.e. early embryonic
primitive neural stem cells and pancreatic multipotent stem cells in the adult. I undertake
3
experiments designed not only to characterize the properties of these novel stem cell
populations, but to trace their lineages either forward or backward in development. In
Chapter 2, I explore the intrinsic default fate of murine embryonic stem cells, which was
found to be that of a primitive neural stem cell. In addition to examining the fundamental
question of how an uncommitted, pluripotent cell will self-organize in the absence of
extrinsic signals, these findings provide insights into the earliest mammalian neural
specification. These findings are used to develop a forward lineage model describing the
ontogeny of embryonic-to-adult neural stem cells. In Chapters 3 and 4, I describe the
discovery and characterization of a novel multipotent adult pancreatic stem cell
population. Aside from demonstrating their characteristics and functional properties, I
undertake experiments aimed at attaining information about the backward lineage, i.e.
embryonic developmental origin, of these exciting stem cells. These distinct populations
provide potential insights into neural and pancreatic development, as well as insights
which may advance therapeutic concerns and possibly disease prevention strategies. In
Chapter 5, I provide a general discussion regarding the findings detailed in Chapters 2-4,
including a commentary regarding the developmental and possible evolutionary
relationships between neural and pancreatic tissues. In order to set the stage and provide a
contextual and conceptual framework for my studies, I take the opportunity in Chapter 1
to present an overview of early embryonic development and lineage-selection events,
followed by an introduction to neural and pancreatic development.
4
Development of the Early Embryo and Embryonic Stem Cells
Genesis of new animal begins at the moment of fertilization, when two very
highly specialized, differentiated cells, i.e. the sperm and the ovum, fuse to yield the
zygote, a totipotent cell responsible for the generation of all cell types within the body, as
well as extraembryonic tissues in utero. While not a stem cell itself, the zygote is the
precursor cell to various stem cell populations which arise and function during
development, and which may persist in the adult, providing a mechanism for continued
cell turnover or regeneration. Intracellular changes induced by fertilization initiate the
subsequent, successive steps of early embryo development – cleavage and gastrulation.
During cleavage, a series of mitotic divisions occur, which parse the enormous
cytoplasmic volume of the zygote into numerous smaller cells. This is followed by
gastrulation, a process in which dramatic rearrangement of relative cell position and
spatial deployment sets up distinct cellular locales. These cellular sub-populations are
specified to generate divergent developmental tissues and organs.
The original cells produced by cleavage are called blastomeres. In most species
(with the exception of mammals), the timing of cell divisions and the relative placement
of blastomeres with respect to one another occur with a stereotypical pattern. This
process is generally under the control of maternally-supplied factors, e.g. proteins and
mRNA, which are stored in the oocyte by the mother prior to fertilization. These
determinants localize to particular regions of the egg, often forming gradients, which set
up distinguishable and functional polarity within the zygote. After fertilization, cell
5
cleavage segregates these determinants such that, after a couple mitotic rounds,
individual blastomeres are no longer equivalent with respect to cellular characteristics
and developmental potential. Thus, differential inheritance of intrinsic factors leads to the
establishment of the first cell lineages within the embryos of most species.
Elucidation of mammalian development, informed primarily through mouse
studies, revealed a different strategy of early embryo lineage specification. The mouse
oocyte does not display any overt polarity, and no factors have yet been described that
exhibit both polarized distribution within the oocyte as well as lineage-specification
capacity. The process of mammalian cleavage also has distinct temporal and spatial
properties. Mitotic cleavages are among the slowest in the animal kingdom, i.e. 12-24
hours apart, and exhibit a unique pattern of the first two cleavage events. The first
division occurs meridionally, however during the second division, one blastomere divides
meridionally while the other does so equatorially, a pattern termed “rotational
cleavage”(Gulyas, 1975). Further, in contrast to most other species, there is mitotic
asynchrony, such that not all blastomeres divide at the same time, often yielding embryos
containing odd numbers of cells. As well, unlike almost all other animal genomes, the
relay of control from the maternal to the zygotic genome occurs quite early during
cleavage, with the zygotic genome being functionally active by the two-cell stage(Piko
and Clegg, 1982;Prather, 1989). During the first two days of development, successive
cleavage rounds produce an eight-cell embryo, termed the morula, in which all of the
blastomeres retain the potential to generate all cell lineages. Moreover, specific ablation
of individual blastomeres at this stage does not preclude development of a normal
6
fetus(Tarkowski, 1959;Tarkowski and Wroblewska, 1967;Tsunoda and McLaren, 1983),
suggesting that early mouse development is governed by a relatively flexible
developmental program. Following the third cleavage round, the eight-cell morula
undergoes another process unique to mammalian embryos – compaction. During
compaction, the previously loosely-associated blastomeres increase cell-cell contact to
form a compact ball of cells. This tightly-packed arrangement is stabilized by tight and
adherens junctions, established by the expression of the cell-adhesion molecule e-
cadherin(Vestweber et al., 1987), forming between cells on the morula surface, while the
inner cells form gap junctions intercellularly. Subsequent rounds of division increase the
topological complexity of the morula, with cells assuming positions either on the outside
or the inside of the morula. Lineage tracing experiments have revealed that the outside
cells, which retain contact with the external environment, become fated to produce the
trophectoderm (TE) cells, which generate the extraembryonic fetal components of the
placenta. The inner cells present at the 16-cell stage, supplemented by the inner daughters
of outer cells produced during the divisions transitioning to the 32-cell stage, form the
inner cell mass (ICM), which is responsible for generating the entire embryo proper as
well as the yolk sac. By the 64-cell stage, the ICM (comprising ~13 cells) and the outer
TE cells have become separate and distinct cell populations, with neither contributing to
the other group, representing the first lineage differentiation event in mammalian
development.
Initially, the morula is a contiguous ball of cells. However, during a process
called cavitation, TE cells secrete fluid into the internal space of the morula, generating a
7
cavity. This cavity is termed the blastocoel, and heralds the blastocyst stage of the
embryo, in which an outer layer of TE cells encases the ICM, located at one pole of the
blastocoelic cavity. Following blastocyst formation, the second lineage decision event
leads to the formation of two morphologically distinct populations from the ICM. The
first is the primitive endoderm (PE), or hypoblast, which forms as a monolayer of cells on
the surface of the ICM facing the blastocoel, and is responsible for generating the
extraembryonic endoderm, which forms the yolk sac. The remaining cells of the ICM are
now termed the epiblast, a population of pluripotent cells responsible for producing all
somatic and germline lineages within the animal. Indeed, studies have shown that
individual epiblast cells can contribute to all of these lineages in chimera
experiments(Gardner and Rossant, 1979;Gardner et al., 1985). Following implantation of
the embryo, gastrulation commences at ~6.5 days post-coitum (dpc). Gastrulation is a
highly-coordinated process involving massive cell movements which yield an embryo,
now termed the gastrula, with multiple tissue germ layers and visible body axes.
Gastrulation begins with a thickening in the posterior region of the epiblast caused by
migration of cells from the lateral regions of the posterior epiblast towards the midline,
followed by movement anteriorly, producing a midline ridge termed the primitive streak.
A depression forms along the length of the primitive streak, termed the primitive groove,
which serves as an opening into the blastocoel. An analogous structure called the
blastopore is present in amphibians. A regional thickening at the anterior end of the
primitive streak comprises the node (Hensen’s node in avians; analogous to the
amphibian blastopore dorsal lip). As soon as the primitive streak is formed, cells begin to
migrate through the groove into the blastocoel to become the endoderm and mesoderm
8
germ layers. Cells passing through the node region enter the blastocoel and migrate
anteriorly to form foregut, head mesoderm, and the notochord. While the presumptive
endodermal and mesodermal precursors are moving inward, the ectodermal precursors
proliferate and remain on the outer surface. These resulting spatial relationships between
tissues make possible the inductive interactions that trigger cellular differentiation and
organ formation (to be revisited below in the context of neural and pancreatic tissue
generation).
The advent of molecular and genetic techniques has allowed us to gain insight
into the mechanisms of the early fate decisions in the developing embryo. As described
above, the first binary fate decision involves the specification of the TE tissue, which is
morphologically distinct from the ICM by the early blastocyst stage. Molecularly, the TE
and the ICM can be distinguished by the expression patterns of several lineage-specific
transcription factors. The TE lineage cells express the gene Cdx2, a homologue of the
Drosophila Caudal, where it functions to promote TE fate(Beck et al., 1995;Strumpf et
al., 2005), whereas the ICM expresses Oct-4, a POU domain protein, and Nanog, a
homeodomain protein, which are associated with pluripotency(Palmieri et al.,
1994;Chambers et al., 2003;Mitsui et al., 2003). Nuclear Oct-4 is detected in all cells of
the early embryo, but its expression becomes restricted to cells if the ICM by the
blastocyst stage(Palmieri et al., 1994). Maternally-supplied Oct-4 transcripts are initially
present, though these decay by the late two-cell stage when control is switched over to
the zygotic genome. Embryos harbouring Oct-4 null mutations display normal
morphological segregation of the TE and ICM lineage compartments, and do implant, but
9
they soon die and lack ICM derivatives(Nichols et al., 1998). This suggests that Oct-4 is
not required for the initial generation of the ICM compartment, but is necessary for
maintaining the pluripotent phenotype and further development of cells therein.
Similarly, Nanog mutant embryos form a morphologically normal blastocyst, though they
die after implantation and lack epiblast derivatives(Mitsui et al., 2003). These two factors
are also key players in conferring pluripotency to embryonic stem (ES) cells (discussed
below). The Cdx2 protein is first detected in certain nuclei at the eight-cell stage, and
becomes restricted to the outer cells in the late morula(Niwa et al., 2005;Strumpf et al.,
2005). Cdx2 mutant embryos also form a blastocyst with normal appearance, although
the ICM markers Oct-4 and Nanog fail to be repressed in the TE, which eventually
collapses and undergoes apoptosis, resulting in embryonic lethality prior to
implantation(Strumpf et al., 2005). It is the interactions between these three factors that
are thought to solidify TE and ICM cell fates.
But how are these two populations of cells with differential gene expression
initially specified from the seemingly homogenous and equipotent early blastomeres?
There exist two primary models regarding this – the “Inside/Outside Model”(Tarkowski
and Wroblewska, 1967) and the “Cell Polarity Model”(Johnson et al., 1981). Both
models are similar in that they depend on differences along the radial axis of the morula
to break the uniformity of the early embryo, rather than differential inheritance of oocyte-
derived cytoplasmic determinants. The “Inside/Outside Model” proposed that cell fate is
established by cell position in the late morula, i.e. whether the cell is an “inside” or
“outside” cell (introduced above). This positional information would then be translated to
10
cell fate in a number of possible ways. The inside and outside microenvironments may
differ chemically, thereby differentially affecting cell fate, or the extent of cell-cell
contact might inform the cell of its position, as the inside cells have symmetrical
neighbouring contacts, whereas the outside cells do not. According to the “Cell Polarity
Model”, cell fate is specified by the eight-cell stage of the morula due to the
establishment of intracellular apical-basal polarity along the radius of the morula during
compaction. This polarity was first observed with the localization of various intracellular
components to the apical (facing outside) pole of outer cells. The angle of division of
subsequent cell replication events then determines daughter cell fates by the symmetric or
asymmetric distribution of “polarity information” during mitosis. Thus, a plane of
division parallel to the morula radius would yield two polarized, similarly-fated cells,
while a plane of division perpendicular to the radius would produce two different cells –
one that retained polarity information and remained on the outer morular surface, and one
apolar cell that was located within. The link between cell polarity and cell fate suggests
that some components of polarity information direct differentiation. It is possible that
apical protein complexes may enhance expression of TE-associated genes, such as Cdx2.
Mutual antagonism between Oct-4 and Cdx2 might then reinforce the segregation of ICM
and TE fates in the blastocyst. This is supported by the findings that Oct-4 and Nanog fail
to be repressed in the TE of Cdx2 mutant embryos(Strumpf et al., 2005), and that the
ICM in some Oct-4 mutants mis-expressed TE markers(Nichols et al., 1998).
Several proteins have been identified that localize to separate cellular pole-
specific complexes of the polarized blastomeres, informed by known apical-basal polarity
11
factors in other systems, such as Drosophila. The apical protein complex contains mouse
homologues of Drosophila Par3 and Par6, as well as the apical membrane protein ezrin
and atypical protein kinase C’s (aPKCs) (Pauken and Capco, 2000;Plusa et al.,
2005;Louvet et al., 1996;Vinot et al., 2005). Proteins localized exclusively to the basal
cell regions include the serine/threonine kinase EMK1 (Par1 homologue) and Lgl (lethal
giant larva homologue)(Vinot et al., 2005). While it is still unclear how these complexes
are initially formed and localized, their distribution is consistent with current models
describing polarity formation in Drosophila(Nelson, 2003;Margolis and Borg, 2005). In
one such model, apical and basal domain segregation is achieved through mutual
antagonism between the respective complexes. The apical complex, containing Par3,
Par6, and aPKC, influences microtubule organization and excludes the basal proteins, e.g.
Lgl, from the apical membrane by direct aPKC-mediated phosphorylation(Plant et al.,
2003;Yamanaka et al., 2003). Whether polarized divisions of the outer cells involve the
segregation and differential inheritance of fate-specifying lineage determinants remains
to be fully elucidated. The specialized apical apparatus could control the orientation of
the mitotic spindle, thereby ensuring the inheritance of localized determinants during
asymmetric divisions, in a manner similar to Drosophila neuroblast lineage
specification(Wodarz, 2005). Currently, there is no direct evidence that Cdx2 protein is
specifically localized to apical regions in the polarized blastomeres, though a recent study
suggested that Cdx2 mRNA might be asymmetrically distributed, leading to later Cdx2
protein production specifically in outside cells(Jedrusik et al., 2008).
12
Insights into the mechanisms governing the second embryonic lineage decision,
the specification of the cells within the ICM to the epiblast and PE lineages, have also
been obtained from modern molecular techniques. Originally, it was thought that all cells
of the ICM were homogenous and equally bipotent, each with the capacity to generate
either epiblast or PE. Cell position was thought to dictate their subsequent lineage
choices, with cells on the blastocoelic surface of the ICM generating PE cells, and cells
of the inner ICM giving rise to epiblast cells by an undefined position-dependent
mechanism. However, it was recently demonstrated that the ICM is not a uniform
population, and that individual ICM cells demonstrate exclusive expression of either
epiblast-specific genes, e.g. Nanog, or PE-specific genes, e.g. Gata-4 and Gata-6, in a
mottled pattern(Chazaud et al., 2006). Lineage-tracing and chimera experiments
confirmed that individual ICM cell progeny are restricted in their fates to one of these
two lineages. Thus, unlike the TE/ICM fate decision, which is largely influenced by cell
position, the epiblast/PE fates appear to be previously established within a mosaic of ICM
cells. These distinctly-fated cells then reorganize to yield the recognizable surface PE and
inner epiblast compartments. Although the details of the upstream mechanisms that give
rise to this mosaicism in epiblast/PE gene expression are unknown, evidence has
accumulated suggesting that signalling through receptor tyrosine kinases via the adaptor
protein Grb2, likely stimulated by fibroblast growth factors (FGFs), establishes the PE
identity by promoting Gata-6 expression(Feldman et al., 1995;Cheng et al.,
1998;Chazaud et al., 2006).
13
Stem cells have been used to study the processes involved with differentiation
during embryogenesis and organogenesis. The embryo, with its developmental plasticity
and high proliferative ability, has traditionally been the preferred source for the isolation
of stem cell populations with varying potencies. Pluripotent cell populations existent
during embryo development have served as a substrate for the derivation of stem cells
that could be maintained in vitro. The most widely-used of these are ES cells, which are
derived from the ICM of blastocysts. Although they are quite proliferative, the ICM cells
are only a transitory pluripotent population in vivo, and thus the ICM cellular constituents
do not display self-renewal in the context of the embryo. However, the stem cell capacity
of the ICM can be revealed by experimental intervention which essentially halts the
progressive differentiation of these pluripotent cells in specific culture conditions. This is
achieved by deriving an outgrowth of cells from the blastocyst, followed by disruption of
the ICM and culture of the disaggregated cells in media containing the cytokine leukemia
inhibitory factor (LIF) (Martin, 1981;Evans and Kaufman, 1981). The ES cell colonies
which arise can be maintained in an undifferentiated state when propagated in the
presence of LIF (provided as a recombinant protein or from culture on mitotically-
inactivated embryonic fibroblasts), and can differentiate with accompanied loss of
pluripotency upon LIF withdrawal. ES cells retain the capacity generate all somatic and
germline lineages in vitro, as well as contributing to these lineages in chimera
experiments(Beddington and Robertson, 1989;Rathjen and Rathjen, 2001;Wobus,
2001;Hubner et al., 2003;Geijsen et al., 2004). Furthermore, individual ES cells are
capable of generating entire, viable mice in vivo using a tetraploid complementation
strategy(Nagy et al., 1993). One of the primary techniques used to evince ES cell
14
pluripotent differentiation in vitro involves the generation of embryoid bodies (EBs),
which are formed by the aggregation of ES cells in the presence of serum and in the
absence of LIF(Desbaillets et al., 2000). During EB formation, its constituent ES cells
differentiate such that the resultant EB contains many different cell types that are fated to
produce multiple cell lineages of all three primary germ layers – the ectoderm, endoderm,
and mesoderm.
The requisite factor for maintenance of mouse ES cells pluripotency in culture is
LIF. In the developing embryo, LIF expression is detected by the late morula stage, but
appears to become localized to cells of the TE, whereas LIF-R expression is observed in
the ICM(Conquet and Brulet, 1990;Nichols et al., 1996). This reciprocal expression
suggests a role for LIF in establishing or maintaining the ICM cell pluripotency, however
neither the absence of LIF(Stewart et al., 1992) or the LIF-R(Li et al., 1995;Ware et al.,
1995) precludes normal embryonic development. Thus, alternative LIF-independent
mechanisms must function to maintain pluripotency of the ICM in vivo. LIF and its
related cytokines signal via the heterodimeric LIF-R/gp130 receptor complex, which
transduces this signal through activation of the JAK/STAT3 and mitogen-associated
protein kinase (MAPK) intracellular pathways(Yoshida et al., 1994;Takahashi-Tezuka et
al., 1998). The activation of STAT3 alone is sufficient for LIF-independent self-renewal
of mouse ES cells in the presence of serum, for which MAPK signalling was also found
to be dispensable(Matsuda et al., 1999). Although native LIF-R signalling does appear to
trigger the MAPK cascade to some degree, singular activation of this pathway appears to
have the predominant effect of promoting ES cell differentiation. In the absence of serum
15
in culture media, LIF alone is insufficient to prevent differentiation of mouse ES cells.
However, with inclusion of bone morphogenetic proteins (BMPs; members of the TGF
superfamily), the ES cell identity can be sustained(Ying et al., 2003a). The BMPs appear
to exert this effect through induction of inhibitor-of-differentiation (Id) protein expression
via the Smad intracellular signalling pathway. Indeed, Id overexpression in the presence
of LIF could maintain ES cells without the need for serum or BMPs. A very recent study
has further advanced our understanding of the factors and pathways involved in ES cell
self-renewal, through developing defined culture media conditions that support ES cell
maintenance without serum or LIF(Ying et al., 2008). The differentiation of ES cells
involves auto-induction of the MAPK pathway by FGF4(Kunath et al., 2007;Stavridis et
al., 2007). However, it was found that neither LIF nor serum/BMPs block this MAPK
signalling(Ying et al., 2003a;Ying et al., 2008), leading to the proposal that these factors
act downstream of phospho-ERK (MAPK pathway downstream signalling component) to
block ES cell lineage commitment. As well, inhibition of glycogen sythase kinase-3
(GSK-3) signalling has been reported to enhance ES cell propagation(Sato et al., 2004).
Thus, a serum- and LIF-free media formulation was developed that contained specific
inhibitors of the FGF-R tyrosine kinases, the MAP kinases, and GSK-3. This inhibitor-
based formulation enabled both the derivation of ES cells, as well as self-renewal with
maintenance of pluripotency(Ying et al., 2008). Thus, it is possible that neutralization of
pro-differentiation signals within the ES cell culture milieu is the key issue for ES cell
propagation, and that BMP/Smad/Id and LIF/STAT3 signalling do not instruct ES cell
self-renewal, but rather function to shield the pluripotent state from differentiation
inductive factors, e.g. MAPK signalling.
16
Progress into our understanding of the pluripotent state has been facilitated by the
identification of a network of auto- and cross-regulatory control mediated by three key
transcription factors, i.e. Oct-4, Nanog, and Sox2, each of which is required for
pluripotency both in vivo and in vitro. Sox2 is expressed in both the ICM and early
primitive ectoderm(Wood and Episkopou, 1999), and Sox2 mutant embryos arrest at a
similar time to Oct-4 and Nanog mutants (described above) (Avilion et al., 2003). Loss of
Oct-4, Sox2, or Nanog in ES cells results in the loss of pluripotency, with associated
spontaneous differentiation along the TE (Oct-4 and Sox2) and PE (Nanog)
lineages(Nichols et al., 1998;Niwa et al., 2000;Chambers et al., 2003;Mitsui et al.,
2003;Chew et al., 2005). Overexpression of Nanog, but not Oct-4 or Sox2, is sufficient to
enable ES cell self-renewal with pluripotency maintenance in the absence of LIF, though
it does not appear that Nanog is a direct target of the LIF/STAT3 pathway(Chambers et
al., 2003). Overexpression of Oct-4 induces ES cell differentiation towards endoderm and
mesoderm (Niwa et al., 2000), while Sox2 overexpressing ES cells differentiate along
neuroectoderm, mesoderm, and trophectoderm lineages(Kopp et al., 2008). Thus, it
appears that levels of Oct-4 and Sox2 must be maintained within a critical range to
support pluripotency. Studies into the function of Sox2 in ES cells have suggested that its
primary function within the pluripotency network is to maintain appropriate levels of
Oct-4 expression(Masui et al., 2007), although an additional role in the initial
establishment of a pluripotency transcriptional network cannot be overlooked(Takahashi
and Yamanaka, 2006;Wernig et al., 2007). Recent work has also drawn into question the
absolute requirement for Nanog in ES cell pluripotency, as Nanog-free ES cells can still
17
be maintained in the pluripotent state, though they do seem more prone to
differentiation(Chambers et al., 2007). Genome-wide mapping of the binding sites of
these three factors have revealed that they bind alone or cooperatively to the promoters of
several hundred target genes(Boyer et al., 2005;Loh et al., 2006). They serve as both
transcriptional activators that enhance expression of genes which maintain pluripotency
(including auto-induction), as well as acting as transcriptional repressors to down-
regulate lineage-specific genes and thereby inhibit differentiation. However, despite our
increased understanding of the signalling pathways involved in ES cell maintenance, e.g.
LIF/STAT3, the mechanism by which these act to maintain the intrinsic pluripotency
transcription factor network remains poorly understood. STAT3 has been shown to
activate certain genes associated with pluripotency, including c-myc(Cartwright et al.,
2005), Nanog(Chambers et al., 2003;Mitsui et al., 2003), eed(Ura et al., 2008),
jmjd1a(Ko et al., 2006), and GABP(Kinoshita et al., 2007), which appears to be
involved in Oct-4 expression maintenance.
In Chapter 2 of this thesis, ES cells are used as an experimental model of the early
pluripotent cells of the ICM to explore the intrinsic default fate program of mammalian
pluripotent cells, and to provide insights into the mechanisms governing the generation of
the earliest neurally-specified cells during development.
18
Neurulation and Neural Induction
The process of embryonic gastrulation produces an embryo with three germ layers
that are positioned to influence one another through inductive interactions. The next
phase of development is organogenesis, in which many organs and organ systems
develop in a simultaneous and coordinated manner. The initiation of the vertebrate
nervous system occurs through a process called neurulation, an early event of
organogenesis. In this section, I will provide a brief general overview of this process,
followed by a discussion of the early neural-inductive events and the mechanisms by
which they occur.
Much of our understanding of neurulation has been obtained from amphibian
embryology studies, which will serve as the bases for the following description of this
process, though it occurs in a similar manner in reptiles, birds, and mammals. Neural
tissue is specified from the ectodermal germ layer, which also gives rise to the epidermis.
During gastrulation, some of the cells migrating through the dorsal lip of the blastopore
(the node in amniotes) move anteriorly to become mesoderm. The dorsal mesoderm
closest to the midline, the chordamesoderm, becomes a rod of connective tissue called the
notochord. The notochord gives structural support to the developing embryo (it is
eventually replaced by the vertebral column), as well as playing a critical role in
distinguishing and pattering the nervous system. Neurulation involves the formation of an
internal neural tube from an external sheet of cells. It is initiated when signals from the
underlying notochordal mesoderm induce a flattening and thickening of overlying
19
ectodermal cells to form an area termed the neural plate. The neural plate lengthens along
the anterior-posterior axis and its lateral edges continue to thicken and elevate to form
ridges or folds. Between these neural folds, a groove forms and deepens as the folds roll
over it to converge at the midline. The folds then fuse at the dorsal midline, forming a
cylinder, the neural tube, and an overlying layer of epidermally-fated ectoderm cells. The
neural tube develops bulges at its anterior end, which subsequently become the major
divisions of the brain (forebrain, midbrain, hindbrain), while the rest of the neural tube
becomes the spinal cord.
The neural crest is a uniquely multipotent and transient population of cells that
originates at the dorsal part of the neural tube through interactions at the junction between
the neuroepithelium and the surface (presumptive epidermal) ectoderm(Dickinson et al.,
1995). Neural crest cells migrate extensively to produce a myriad of differentiated cell
types, including neurons and glia of the peripheral nervous system, adrenomedullary
cells, melanocytes, and many of the skeletal and connective tissue components of the
head(Dupin et al., 2006).
Neural induction is the developmental event when ectodermal cells become first
specified as neural precursor cells(Wilson and Edlund, 2001). Later in development,
these specified cells become committed to the neural fate, and will no longer respond to
signals instructing alternate fates. Pioneering studies into the mechanisms of neural
induction were performed in the mid-1920s by Spemann and Mangold. Using amphibian
embryos, it was shown that transplantation of the dorsal-most lip of the blastopore to the
20
ventral side (prospective belly) of another embryo at the gastrula stage led to the
development of a second body axis(Spemann and Mangold, 1924;Spemann and Mangold,
2001). Notably, the induced second central nervous system was formed by the host
ventral ectoderm that would have been fated towards epidermis. Because of these
findings, Spemann referred to the dorsal lip and its derivates as the “organizer”, which
was responsible for providing the signals that instructed proximal ectoderm to adopt a
neural fate. Soon thereafter, equivalent structures were identified in other vertebrates: the
“shield” in teleosts(Oppenheimer, 1936b), and (Henson’s) node in birds and
mammals(Waddington, 1932;Waddington, 1933;Waddington, 1936;Waddington, 1937).
Such regions were also demonstrated to have neural plate inductive activity(Waddington,
1934;Oppenheimer, 1936a;Shih and Fraser, 1996). Interestingly, it was found that they
did so not only when transplanted into another individual of the same species, but also
when transplanted across classes(Waddington, 1934;Kintner and Dodd, 1991;Hatta and
Takahashi, 1996), suggesting that the mechanisms governing neural induction are
conserved throughout vertebrate species.
By what mechanim(s) does the organizer/node region instruct the neural fate in
adjacent ectodermal cells? Initially, the experiments described above led to the proposal
that the organizer was responsible for providing a positive instructive signal which
directed the unspecified ectoderm towards the neural lineage, and in the absence of this
signal, the ectoderm would proceed towards epidermal fates. For more than sixty years,
many studies were performed trying to elucidate the molecular identity of the organizer-
derived neuralizing signal, with the hope of discovering a “master neural inducer”,
21
though these met with little success. More recent studies have generated findings which
challenged the validity of the mechanism proposed in this “classical” model. First, it was
observed that dissociation of amphibian ectodermal animal cap explants, followed by
culture in the absence of organizer tissues, resulted in their differentiation to neural cells,
whereas undissociated explants formed epidermis(Grunz and Tacke, 1989;Sato and
Sargent, 1989;Godsave and Slack, 1991). Then, a second clue was obtained from
experiments initially designed to study the mechanisms of mesoderm
formation(Hemmati-Brivanlou and Melton, 1992). It was found that undissociated
ectodermal explants expressing a dominant-negative receptor for activin (a member of
the TGF superfamily of growth factors) became neural when cultured(Hemmati-
Brivanlou and Melton, 1994). It was later shown that this activin-receptor dominant-
negative effectively inhibited signalling by multiple TGF-related molecules(Schulte-
Merker et al., 1994;Hemmati-Brivanlou and Thomsen, 1995), and a similar neuralizing
activity was observed with dominant-negative forms of the BMP receptor(Xu et al.,
1995;Hawley et al., 1995). These findings led to the idea that neural tissue might be
induced by the removal of some unknown inhibitory factor(s). Soon, several genes
encoding proteins with potent neuralizing activity were identified as being expressed by
the organizer, i.e. Noggin, Chordin, and Follistatin(Lamb et al., 1993;Smith et al.,
1993;Sasai et al., 1995;Hemmati-Brivanlou et al., 1994). While such factors were initially
thought to represent the instructive neuralizing signal from the organizer, subsequent
insights into their mechanism of action were not entirely consistent with the existent
positive induction model. It turned out that these factors were extracellular binding
partners of BMPs, and that their neuralizing ability depended on this inhibitory
22
interaction with the BMPs(Piccolo et al., 1996;Zimmerman et al., 1996;Fainsod et al.,
1997). Thus, the mechanism of action of the secreted organizer-derived molecules
appeared to be through interference with the binding of BMPs to their affiliated receptors
on ectodermal cells. Indeed, it was further found that BMPs exerted potent anti-
neuralizing activity, while promoting epidermal differentiation, even in dissociated
cells(Hawley et al., 1995;Wilson and Hemmati-Brivanlou, 1995). As well, neuralization
was inhibited after dissociation of animal caps obtained from embryos that were
previously injected with RNA encoding effectors of BMP4 signalling, i.e. Msx1, Smad1,
and Smad5(Suzuki et al., 1997a;Suzuki et al., 1997b;Wilson et al., 1997), consistent with
the view that neural fates are inhibited by BMP activity. Moreover, the expression pattern
of BMP4 conforms to its proposed anti-neural function, as it is widely expressed
throughout the entire ectoderm in the early gastrula, and subsequently disappears from
the prospective neural plate when the organizer appears(Fainsod et al., 1994). BMP
proteins feed back to stimulate their own transcription(Biehs et al., 1996), and so BMP
protein antagonism in the vicinity of the organizer explains the local disappearance of
BMP expression. Additional secreted BMP-antagonizing molecules that are expressed in
the organizer region have been identified, such as Cereberus(Bouwmeester et al.,
1996;Belo et al., 1997), Gremlin, Dan, and Drm(Hsu et al., 1998;Pearce et al.,
1999;Dionne et al., 2001), and Ogon/Sizzled(Wagner and Mullins, 2002;Yabe et al.,
2003). In the mouse, loss of function of individual BMP antagonists Noggin, Chordin,
Follistatin, or Cerberus1 do not result in dramatic defects in neural induction (Matzuk et
al., 1995;McMahon et al., 1998;Simpson et al., 1999;Bachiller et al., 2000), likely due to
functional redundancy between various BMP inhibitors. However, mice with a double-
23
knockout for Noggin and Chordin display severe defects in forebrain and head
development(Bachiller et al., 2000). As well, a Xenopus study using simultaneous
depletion of Noggin, Chordin, and Follistatin with morpholino oligonucleotides
demonstrated an almost complete loss of the neural plate(Khokha et al., 2005). Overall,
these findings led to the development of the current, and widely-accepted “default”
model, which states that within the ectodermal germ layer, each individual cell has an
intrinsic and autonomous program to become a neural cell(Hemmati-Brivanlou and
Melton, 1997;Munoz-Sanjuan and Brivanlou, 2002). In the context of the developing
embryo, ubiquitous BMP expression is actively suppressing the manifestation of this
default neural fate. Thus, the organizer/node tissue does not serve to provide a positive
instructive signal, but rather secretes factors which antagonize BMP signalling, thereby
dis-inhibiting the default neural program in proximal ectodermal cells.
Although it is the prevailing paradigm, there exist challenges to the default model.
While the role of the BMP pathway in determination of ectodermal fates is well
established, an issue of contention is whether BMP inhibition alone is sufficient for
neural induction, or whether other signals play an independent or cooperative role. If any
of these accessory signals are required in an instructive capacity, then the neural fate
cannot be said to truly represent the default. The strongest challenges to the default model
come from studies in the chick, in which mis-expression of BMP antagonists in
competent epiblast did not induce the expression of neural markers, and that a grafted
source of BMP protein did not inhibit neural plate development(Streit et al., 1998;Streit
and Stern, 1999;Linker and Stern, 2004). These studies suggested that BMP inhibition
24
alone is insufficient for neural induction, however it is uncertain how complete the
effective BMP inhibition was in these experiments, and it is possible that individual
BMPs escaped (or experienced insufficient) inhibition and were allowed to prevent
neural fate induction. As well, signalling by the FGFs and Wnts has been suggested to
play an instructive role in neural tissue specification in several vertebrates(Baker et al.,
1999;Streit et al., 2000;Wilson et al., 2000;Wilson et al., 2001). However, it is unclear
whether these factors are required for the initial neural fate induction, or for subsequent
maintenance and expansion of a neurally-specified cell population. Furthermore, their
mode of action may be primarily via modulation of BMP expression at the transcriptional
level(Bainter et al., 2001), consistent with a BMP-inhibition mediated neural induction
mechanism.
In the mouse, the node region may not be solely responsible for all neural tissue
induction. A distinct cell population, the anterior visceral endoderm (AVE), develops
from the extraembryonic PE, and migrates during early development to lie beneath the
anterior end of the epiblast(Beddington and Robertson, 1998;Beddington and Robertson,
1999). The AVE is required for anterior neural tissue and head development, but not for
generation of more posterior central nervous system components, and thus may represent
a separate “head organizer”, a concept originally proposed by Spemann(Spemann, 1931).
This proposal was supported by the findings that in mice lacking a node and its
derivatives, e.g. in mutants for the transcription factor HNF3, the neural plate and
anterior neural derivatives still form(Ang and Rossant, 1994), and mouse embryos
lacking the AVE did not develop head structures(Thomas and Beddington, 1996).
25
Molecularly, the AVE expresses Lefty, an inhibitor of Nodal (a TGF superfamily
member), and Cerberus, which may underlie its role in neural tissue specification(Belo et
al., 1997). However, it was discovered that the AVE alone is insufficient to induce neural
fates in total epiblast explants(Kimura et al., 2000), or in transplantation
experiments(Tam and Steiner, 1999). Further, neural-inducing activity of the AVE was
only found when it was grafted in combination with prospective organizer tissue and the
appropriate responding tissue (future forebrain)(Tam and Steiner, 1999). This suggested
that the AVE plays only an indirect or permissive role in neural induction.
Brain Development and Neural Stem Cells
The original neural tube is composed of germinal neuroepithelium that is one cell
layer thick, containing highly proliferative neural precursor cells that express the neural
precursor markers Sox1(Pevny et al., 1998) and Nestin(Lendahl et al., 1990;Dahlstrand et
al., 1995). The differentiation of the neural tube into different brain regions occurs in
three different ways. On the gross anatomical level, the neural tube and its lumen bulge
and constrict to form the chambers of the brain and the spinal cord. At the tissue level,
the cell populations within the wall of the neural tube rearrange themselves to form the
different functional regions of the brain and the spinal cord. Finally, on the cellular level,
the neuroepithelial cells themselves divide and differentiate into the numerous types of
neurons and glial cells.
increasing their numbers. As development proceeds, these cells undergo some changes in
their gene expression patterns, cytological characteristics, and differentiation potential.
With the onset of neurogenesis, neuroepithelial cells switch to an asymmetric mode of
division, generating distinct types of secondary neural precursor cells (radial glial cells,
basal progenitors), and neurons. The secondary neural precursor cells also undergo both
symmetric and asymmetric types of division. In general, neuronogenesis occurs first,
followed by gliogenesis. With the generation of neurons, the neuroepithelium is
transformed into a tissue with multiple layers, and the layer that lines the ventricle (the
most apical cell layer that contains most of the precursor cell bodies) is referred to as the
germinal ventricular zone (VZ). These ventricular neuroepithelial precursors give rise to
cells with astroglial features, the radial glia cells, from which most of the neurons of the
brain are derived(Hartfuss et al., 2001;Anthony et al., 2004). The radial glia contact the
inner ventricular surface and the outer pial surface of the neural tube, guiding neuronal
migration away from the VZ. The basal progenitors are distinguished from the radial glia
by the fact that their nuclei undergo mitosis at the basal side of the VZ, though they
originate from divisions of cells at the apical surface(Smart, 1973;Haubensak et al.,
2004;Noctor et al., 2004). The basal progenitors contribute to neurogenesis by
undergoing symmetric cell divisions that generate two neuronal daughter cells, thereby
functioning to increase the number of neurons generated from a given number of apical
precursors by allowing another round of division distant from the apical surface. When
early neuroblast formation has ceased, the remaining neuroepithelial cells in the VZ
begin to differentiate into glioblasts. Neuronal birth initiates at about embryonic day (E)
27
10, and persists in certain brain regions throughout adulthood. In the early post-natal
brain, significant neurogenesis remains in three regions – the cerebellum, the dentate
gyrus (DG) of the hippocampus, and the olfactory bulb. The cerebellar external granule
layer contains neurogenic precusors that are actively generating neurons during the first
few post-natal days, while neurogenesis persists in the DG and the olfactory bulb
throughout adult life.
Neural stem cells (NSCs) are thought to be responsible for the sustained
neurogenic potential within the adult brain. The NSC population expresses the glial
marker GFAP, and resides in the subependymal layer of the lateral subventricular zone
(SVZ)(Doetsch et al., 1999;Morshead et al., 2003). Neural progenitors derived from
asymmetric divisions of SVZ NSCs migrate along the rostral migratory stream to take up
residence within the olfactory bulb, where they differentiate into granule and
periglomerular cell interneurons(Lois and Alvarez-Buylla, 1994;Luskin, 1993). Such
subependymal NSCs have been described not only in mouse, but in both monkey and
human primates(McDermott and Lantos, 1990;Kornack and Rakic, 2001;Pincus et al.,
1998;Kukekov et al., 1999). SVZ neurogenesis is modulated by the olfactory experience
of animals(Lledo and Saghatelyan, 2005). Deprivation of olfactory inputs hinders the
maturation and survival of newborn neurons in the olfactory bulb, and interference with
neuroblast migration to the olfactory bulb causes deficits in olfactory
discrimination(Gheusi et al., 2000). Conversely, enriched odour exposure increases the
survival and integration of newborn neurons and transiently improves odour
memory(Rochefort et al., 2002). Pregnancy also enhances olfactory bulb neurogenesis, an
28
effect mediated by prolactin, which may serve a key function, as olfactory discrimination
is critical for recognition and rearing of offspring(Shingo et al., 2003).
In the DG of the hippocampus, newborn neurons migrate into the granule cell
layer and integrate as hippocampal granule cells(Cameron et al., 1993;Kuhn et al., 1996).
Aside from the rodent, hippocampal neurogenesis has also been observed in both monkey
and human primates(Gould et al., 1999b;Kornack and Rakic, 1999;Eriksson et al., 1998).
Hippocampal neurogenesis can be modulated by many different factors. Some of these
which enhance neurogenesis are environmental enrichment(Kempermann et al., 1997),
increased motor activities(van Praag et al., 1999), electroconvulsive and other seizure
models(Scott et al., 1998;Madsen et al., 2000), antidepressant treatment(Malberg et al.,
2000), and estrogen or IGF-1 administration(Tanapat et al., 1999;Aberg et al., 2000).
Two major negative regulators of hippocampal neurogenesis are aging and stress(Kuhn et
al., 1996;Gould et al., 1997;Gould et al., 1998). Associative learning tasks have been
shown to enhance the generation and survival of new hippocampal DG neurons(Gould et
al., 1999a), suggestive of a role for hippocampal neurogenesis in cognition and learning.
This has been supported by the findings that interference with, or ablation of,
neurogenesis led to impairment of trace memory formation and learning in contextual
fear conditioning tasks(Shors et al., 2001;Saxe et al., 2006;Winocur et al., 2006).
Although it sustains adult neurogenesis, the provenance of new DG neurons remains
controversial. Original reports using in vitro assays suggested the presence of intra-
hippocampal precursors with stem cell properties(Palmer et al., 1997), however it was not
demonstrated explicitly that these were resident within the DG. Indeed, a more recent
29
study has directly examined this issue, and concluded that the precursors within the DG
represent restricted progenitors, while bona fide NSCs are located in the adjacent SVZ
region(Seaberg and van Der Kooy, 2002).
Thus, NSCs resident within the SVZ region represent the true stem cells of the
adult brain. One of the primary techniques used to study and assess the properties of these
NSCs has been an in vitro growth factor-dependent clonal colony formation and
differentiation assay, the “neurosphere” assay, which provides information about both
cardinal stem cell properties, i.e. self-renewal and multipotentiality. Using such assays,
the presence of NSCs has been demonstrated from embryonic periods through
adulthood(Reynolds and Weiss, 1992;Morshead et al., 1994;Tropepe et al., 1997;Tropepe
et al., 1999). A population of NSCs that are proliferatively responsive to FGF2 was
identified as early as E8.5, while a later-emerging, yet lineage-related, EGF-responsive
population was present by E14.5 in mice. In spite of this, a comprehensive understanding
of NSC ontogeny has not been fully elucidated. In Chapter 2 of this thesis, I utilize ES
cells to explore the earliest neural precursor generation, with the resultant discovery of
primitive NSCs (a fate found to be the default of pluripotent ES cells). I further develop
an ES cell-based model describing the ontogeny of NSCs. In Chapter 3 of this thesis,
adult NSCs are utilized as a comparator to a novel population of adult multipotent
pancreatic precursor cells.
Pancreatic Development, Regeneration, and Adult Pancreatic Precursor Cells
The pancreas is a heterogeneous organ that serves two major functions. First, it is
responsible for the production of digestive enzymes, e.g. trypsin and amylase, which are
produced by the exocrine acinar cells, which comprise over 95% of the pancreatic mass.
These enzymes are secreted into the acinar lumens, which drain into a highly branched
system of ducts which eventually feed into the common bile duct. Second, it is
responsible for the tightly-regulated control of glucose homeostasis, despite large
fluctuations in glucose delivery or removal from the circulation. This is achieved by the
endocrine cells contained within the islets of Langerhans, pseudo-spherical islands
embedded within the exocrine tissue of the pancreas. Insulin-producing cells are the
most prominent endocrine cell type (comprising 60-80% of islet cells), followed by
glucagon-producing cells. The remaining islet cells include cells, which produce
somatostatin, PP cells, which produce pancreatic polypeptide, and cells, producing
ghrelin. The endocrine cells achieve glycemic control by coordinating the disposition of
nutrient input from meals as well as the flow of endogenous substrates(Cherrington and
Vranic, 1971;Amir and Shechter, 1988;Levine, 1982;Saltiel and Kahn, 2001).
Endogenous glucose production occurs primarily in the liver, while insulin-sensitive
glucose uptake and storage occurs mainly in the liver, muscle, and adipose tissue. Insulin
is a strong anabolic hormone, promoting storage of carbohydrate, fat, and protein by
stimulating glycogenesis, lipogenesis, and protein synthesis, while inhibiting
glycogenolysis, lipolysis, and protein breakdown. Following the ingestion of a glucose
load, normal glucose homeostasis is principally maintained by the actions of insulin,
31
which is secreted from the cells in response to an increase in plasma glucose
concentration. The rise in insulin levels suppresses endogenous glucose output and
increases storage of glucose as glycogen in the liver. The elevation of insulin levels also
stimulates the uptake of glucose by peripheral tissues, i.e. muscle and adipose tissue.
These effects serve to lower plasma glucose levels, thereby removing the stimulus for
further insulin release. Hence, insulin acts via a negative feedback regulatory loop to
control plasma glucose levels.
The predominant diseases of the pancreas are the diabetes mellitus (DM) group of
metabolic diseases, characterized by hyperglycemia resulting from defects in insulin
secretion, insulin action, or both. According to the 2007 Diabetes Atlas published by the
International Diabetes Federation, diabetes affected more than 240 million people
worldwide, with a projected incidence of 380 million by the year 2025. Diabetes is
classified into two broad categories(Gavin, 2003). Type 1 DM, previously called insulin-
dependent DM (IDDM) or juvenile-onset diabetes, accounts for less than 10% of all
cases, and is characterized by immune-mediated destruction of the cells resulting in
absolute insulin deficiency(Atkinson and Eisenbarth, 2001). This autoimmune condition
has multiple genetic predispositions and is also related to environmental factors that are
still poorly defined. Type 2 DM, previously called non-insulin-dependent DM (NIDDM)
or adult-onset DM, accounts for greater than 90% of all cases, and is characterized by
peripheral insulin resistance and relative insulin deficiency(Edelman, 1998). Insulin
resistance manifests as inappropriate glucose output and deficient glucose storage in the
liver, and impaired glucose uptake and storage in muscle and adipose tissues. The relative
32
insulin deficiency is primarily due to inadequate and dysfunctional insulin secretory
responses of the cells to a glucose stimulus. Extreme acute hyperglycemia can cause
severe life-threatening health problems, and chronic hyperglycemia is associated with a
variety of long-term complications including macrovascular diseases (e.g. ischemic heart
disease, stroke, gangrene), microvascular diseases (e.g. retinopathy, neuropathy,
nephropathy), and others that reduce life expectancy(Nathan, 1993). As such, much work
has been focussed on the development of strategies to combat these diseases, including
the search for pancreatic precursor cells which may serve as a cellular source for the
production of new cells to replace those that are lost or dysfunctional. In the remainder
of this section, I will provide a brief overview of pancreatic development, followed by a
concise introduction to pancreatic regenerative potential and putative pancreatic
precursors.
Developmentally, the pancreas arises from the endoderm germ layer. The first
morphologic evidence of the pancreas occurs at about E9.5 in the mouse with a
condensation of the mesenchyme overlying the dorsal aspect of the endodermal gut tube,
just distal to the stomach. The underlying endoderm then evaginates into the overlying
mesenchyme forming the dorsal pancreatic bud. At this time, the notochord is embedded
in the endoderm, and it is through inductive and permissive interactions that the
prospective dorsal pancreatic endoderm becomes specified. It was found that notochord-
derived signals (i.e. Activin-B, a member of the TGF superfamily, and FGF2) were
responsible for specifically repressing Sonic Hedgehog (Shh) expression in the localized
prospective pancreatic endoderm(Kim et al., 1997;Hebrok et al., 1998). A similar, ventral
33
pancreatic bud begins to arise adjacent to the hepatic diverticulum approximately 12
hours later, though it does so without interaction with the notochord, and is specified by
signals from the overlying cardiogenic mesenchyme. Subsequently, as the developing
stomach and duodenum rotate (at about E12-13), the dorsal and ventral buds come into
contact with each other and fuse to form the definitive pancreas. At about E13-14,
dramatic changes occur in the cellular architecture of the pancreas, involving a major
amplification of endocrine cells, particularly cells. Coincidently, rapid branching
morphogenesis and acinar cell differentiation occurs. The endocrine cells coalesce into
islets only later during gestation, at about E19.
The entire early pancreatic rudiments and portions of the surrounding gut tube
express the homeobox transcription factor PDX-1, which can be detected as early as
E8.5-9 in the presumptive pancreatic endoderm before it has visibly thickened (Guz et al.,
1995). PDX-1 is thought of as a “master” pancreatic gene, as when it is removed from
mice by targeted mutagenesis or from humans by a naturally occurring mutation, the
pancreas fails to form(Jonsson et al., 1994;Stoffers et al., 1997). Both the endocrine and
the exocrine portions of the pancreas have been shown to arise from PDX-1-expressing
precursors in genetic lineage-tracing experiments(Gu et al., 2002). Another transcription
factor, Ptf1a, is also expressed in the early pancreas, and its expression remains pancreas-
specific throughout development(Kawaguchi et al., 2002). Lineage-tracing studies
revealed that Ptf1a-expressing precursors also contribute to all mature pancreatic
lineages, although its expression becomes restricted to acinar precursors by
approximately E13.5. Consistent with these observation, Ptf1a was found to activate
34
acinar-specific genes(Krapp et al., 1996), and Ptf1a-deficient pancreata completely lack
acinar cells(Krapp et al., 1998). PDX-1, meanwhile, was found to activate the rodent
Insulin-1 gene, and from E15.5 onwards its expression becomes primarily restricted to
cells(Ohlsson et al., 1993). The transition to restricted expression of PDX-1 and Ptf1a
generally coincides with the conversion of precursors into mature endocrine and exocrine
cells. PDX-1-expressing cells give rise to cells expressing Neurogenin-3 (Ngn3), which
currently sits atop the pancreatic endocrine lineage transcription factor hierarchy(Gu et
al., 2002). The initial segregation of Ngn3-expressing cells appears to be under the
control of Notch signalling. Mice with null mutations for a key Notch ligand present in
the developing pancreas, Delta-like-1 (Dll1), or for the transcription factor RBP-Jk, a
mediator of Notch signalling, both demonstrated an accelerated and over-abundant
commitment of the early pancreatic epithelium to the endocrine lineage(Apelqvist et al.,
1999). These data, along with other studies examining Notch signalling downstream
components as well as Ptfa activity and Ngn3 expression profiles(Jensen et al.,
2000;Murtaugh et al., 2003;Esni et al., 2004a), indicate that Notch signalling in the early
developing pancreas is responsible for maintaining the undifferentiated state of pancreatic
precursors by repressing exocrine and endocrine differentiation through inhibition of
Ptf1a activity and Ngn3 expression, respectively.
Ngn3 expression can be first detected in the early pancreatic epithelium at E9,
followed by escalation of expression peaking at about E15.5, with a subsequent decline
such that expression within adult islet cells is very rare(Gradwohl et al.,
2000;Schwitzgebel et al., 2000). Mice lacking Ngn3 fail to develop any pancreatic
35
endocrine cells(Gradwohl et al., 2000), and mice with transgenic Ngn3 expression under
control of the PDX-1 promoter display a massive conversion of pancreatic cells into
endocrine cells(Apelqvist et al., 1999). As well, mis-expression of Ngn3 in pancreatic
ductal cells of both mouse(Gasa et al., 2004;Mellitzer et al., 2006) and human(Heremans
et al., 2002) was found to induce an endocrine program. Ngn3 activates a battery of
transcription factors that constitute a core program of endocrine development. These
include Islet-1, NeuroD, and Insm1, as well as downstream genes involved in specifying
the various endocrine cells fates, such as Pax6, Pax4, Arx, Nkx2.2, Nkx6.1, MafA, and
HlxB9(Huang et al., 2000;Marsich et al., 2003;Smith et al., 2003;Collombat et al.,
2003;Watada et al., 2003;Pedersen et al., 2005;Raum et al., 2006;Jensen et al.,
2000;Ahlgren et al., 1997;Olbrot et al., 2002). Refer to Figure 1.1 for a simplified
schematic overview of their involvement in pancreatic cell subtype specification.
It is now well-accepted that the cell mass expands postnatally and that the
pancreas retains some degree of regenerative capacity, although the source of new
cells, especially in the adult, remains controversial. Throughout life, the cell mass is
dynamic and undergoes compensatory changes to meet demand. In both rodents and
humans, the cell mass expands during normal postnatal development and into
adulthood, correlating with body mass(Montanya et al., 2000;Finegood et al.,
1995;Bonner-Weir, 2000). As well, various physiological conditions and experimental
manipulations are associated with an increase in cell mass. During pregnancy in the rat,
the cell mass expands by 50%(Parsons et al., 1992). Experimental manipulations found
to increase cell mass or to induce neogenesis include a transgenic mouse model of
Foregut Endoderm
36
Simon
1989), dietary treatment with soybean trypsin inhibitor(Weaver et al., 1985),
overexpression of interferon-gamma (IFN) in cells(Sarvetnick et al., 1990), partial
pancreatectomy(Bonner-Weir et al., 1993), cellophane wrapping of the head of the
pancreas(Rosenberg et al., 1983), Glucagon-like peptide-1 (GLP-1)/exendin-4
treatment(Xu et al., 1999), and after ligation of the main pancreatic duct(Walker et al.,
1992). However, despite all of these conditions with adult cell neogenesis, the cellular
source of the nascent cells remains unclear. It is now known that cells do retain a
limited capacity for replication(Messier and LeBlond, 1960;Kassem et al., 2000),
however this process is unlikely to account for all new cell production. Several groups
have suggested the existence of intra-pancreatic precursor cells which could be called
upon to maintain cell levels in the adult. It has been suggested that adult pancreatic
Nestin-expressing cells represent functional precursors in both rodent and human
tissue(Zulewski et al., 2001). However, this proposition remains controversial(Piper et
al., 2002;Treutelaar et al., 2003;Street et al., 2004), and so while Nestin may mark some
proliferative pancreatic precursors in vitro it is unlikely to be a specific marker for
precursors in vivo (discussed further in Chapter 3 of this thesis). Another location
proposed to house pancreatic precursors is the pancreatic ducts, supported by the
observation that in the IFN mouse model there is continual destruction of islets, with
continual proliferation of the ductal epithelium and formation of new islets(Gu and
Sarvetnick, 1993). Further, it was shown in vitro that some cultured human ductal cells
appeared to generate islet-like structures(Bonner-Weir et al., 2000). The pancreatic stem
cell field was disjarred recently by a notable study which argued that all new cells in
38
the adult were formed exclusively by replication of existing cells, and that there was no
evidence to suggest the existence of, and a role for, pancreatic stem cells(Dor et al.,
2004). This group also performed a follow-up study suggesting that there were no
pancreatic stem cells in the developing embryo nor the adult, and that the pancreatic
organ is formed during development by progenitors that are autonomously restricted,
capable of producing only a fixed amount of tissue(Stanger et al., 2007). These studies
generated a fair bit of controversy within the field, though they are not without criticism
and alternative interpretation of their data (discussed in more detail in Chapter 4 of this
thesis).
In Chapters 3 and 4 of this thesis, I describe the discovery and characterization of
a novel population of pancreatic precursors resident within the adult mammalian
pancreas. This characterization includes a demonstration of in vitro proliferative capacity,
self-renewal, and differentiation potential, as well as in vivo data regarding the cellular
identity, developmental origin, and functionality within the adult pancreas. Overall, my
data supports the existence of an insulin-expressing intra-pancreatic stem cell that is
multipotential in the pancreatic and neural lineages, and which manifests this potential in
vivo during normal adult pancreatic cell turnover. These findings may yield insights into
normal pancreatic development and tissue homeostasis, as well as providing a cellular
substrate for the ex vivo generation of islet cells for therapeutic considerations.
39
Content within this chapter has been published as:
Smukler SR, Runciman SB, Xu S, van der Kooy D. Embryonic stem cells assume a
primitive neural stem cell fate in the absence of extrinsic influences. J Cell Biol. 2006
Jan 2;172(1):79-90
40
SUMMARY
The mechanisms governing the emergence of the earliest mammalian neural cells
during development remain incompletely characterized. A default mechanism has been
suggested to underlie neural fate acquisition, however an instructive process has also
been proposed. We utilized mouse ES cells to explore the fundamental issue of how an
uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic
signals, and what cellular fate will result. To assess this default state, ES cells were
placed in conditions that minimize external influences. Individual ES cells were found to
rapidly transition directly into neural cells, a process shown to be independent of
suggested instructive factors (e.g. FGFs). Further, we provide evidence that the default
neural identity is that of a primitive neural stem cell (NSC). The exiguous conditions
used to reveal the default state were found to present primitive NSCs with a survival
challenge (limiting their persistence and proliferation), which could be mitigated by
survival factors or genetic interference with apoptosis.
41
INTRODUCTION
The emergence of the earliest neural cells during mammalian development, and
the mechanisms which govern this process, remain incompletely characterized. Such cells
are likely to be neural precursors or stem cells, though the ontogeny of the neural stem
cell (NSC), which can be isolated from embryonic and adult forebrain (Weiss et al.,
1996;Gage, 2000), has not been fully elucidated. During development, neural cells arise
from the ectodermal germ layer, which also produces epidermis. According to the
classical model of this process, conceptualized largely from amphibian embryology
studies, nascent embryonic ectoderm receives a positive signal from a specialized group
of dorsal mesodermal cells, termed the “organizer”, which instructs the adjacent
ectodermal cells to adopt a neural fate (Harland and Gerhart, 1997;Weinstein and
Hemmati-Brivanlou, 1999;Spemann and Mangold, 2001). The structural equivalent of the
organizer in amniotes is the “node”. It was thought that organizer/node-derived signals
were necessary for the process of neural induction, and that in their absence the ectoderm
would adopt an epidermal fate.
More recent data have challenged the validity of this classical model. Low-density
cultures of dissociated ectodermal cells, in the absence of organizer tissue, were found to
differentiate into neural cells (Sato and Sargent, 1989;Godsave and Slack, 1991;Grunz
and Tacke, 1989). Furthermore, undissociated ectodermal explants expressing a
dominant-negative receptor for activin (a member of the TGF family of growth factors),
which effectively inhibited signalling of multiple TGF-related molecules (Schulte-
42
Merker et al., 1994;Hemmati-Brivanlou and Thomsen, 1995), were shown to become
neural when cultured in vitro (Hemmati-Brivanlou and Melton, 1994). Signalling
molecules secreted from the organizer tissue, such as Noggin, Chordin, and Follistatin,
were found to exert potent neuralizing effects (Lamb et al., 1993;Sasai et al.,
1995;Hemmati-Brivanlou et al., 1994), and thus were initially thought to represent the
instructive neuralizing signal. However, the mechanism by which they promoted neural
differentiation of ectodermal cells was not entirely consistent with the existing positive
induction model. The neuralizing effects of these factors were found to depend on
inhibitory interactions with bone morphogenic proteins (BMPs), members or the TGF
family of molecules, which strongly inhibit neural differentiation (Zimmerman et al.,
1996;Piccolo et al., 1996;Fainsod et al., 1997). Thus, their mechanism of action appeared
to be through prevention of BMP binding to their cognate receptors on ectodermal cells.
These findings led to the development of the currently more widely accepted model, the
“default model”, which states that each individual ectodermal cell has an intrinsic default
program to become a neural cell (Munoz-Sanjuan and Brivanlou, 2002). In the context of
the intact embryo, this default program is being actively suppressed by ubiquitously
expressed BMPs. Thus, the organizer tissue does not provide a positive inductive signal,
but rather secretes factors that antagonize BMP signalling, thereby dis-inhibiting the
default neural program in proximal ectodermal cells.
Several subsequent studies have challenged the default model of neural fate
acquisition. For example, experiments in chick have suggested that BMP inhibition may
not be sufficient to induce neuralization (Streit et al., 2000;Linker and Stern, 2004).
43
However, it is uncertain how complete the BMP inhibition was in these studies, and it is
possible that the activity of individual BMPs was insufficiently suppressed to allow
neuralization to occur (and/or that some BMP subtypes or other neural inhibitors escaped
blockade). It has also been suggested that other factors, such as FGF and Wnt signalling,
are involved with neural specification in several vertebrates (Baker et al., 1999;Wilson et
al., 2000;Wilson et al., 2001;Streit et al., 2000), though it is currently unresolved as to
whether they are required for the initial neural fate change or for the later expansion of
this neural population. Further, their mechanism of action may be through modulation of
BMP gene transcription (Bainter et al., 2001), consistent with a model of BMP-inhibition
mediated neuralization.
There are currently few published studies examining the neural default model in
mammalian cells, and there is controversy over whether such a default neural mechanism
exists in mammals. In an effort to determine whether a default mechanism underlies
neural fate specification from uncommitted mammalian precursors, we undertook studies
utilizing mouse embryonic stem (ES) cells, which are derived from the inner cell mass
(ICM) of the blastocyst-stage embryo, and represent a model of the earliest pluripotent
mammalian cell (Martin, 1981;Evans and Kaufman, 1981). ES cells are capable of
generating entire, viable mice in vivo (Nagy et al., 1993), and are able to produce most, if
not all, cell types in vitro (Beddington and Robertson, 1989;Rathjen and Rathjen,
2001;Wobus, 2001). Use of ES cells to investigate neural determination can potentially
provide many insights into the developmental process. Importantly, though, their use in
assessing a default fate specification mechanism allows us to explore a more basic and
44
fundamental issue, i.e. how an uncommitted, pluripotent mammalian cell will self-
organize in the absence of extrinsic instructive or inhibitory signals, and what cellular
configuration/fate will result.
The standard methodology for the in vitro differentiation of ES cells typically
involves the formation of embryoid bodies (EBs) (Desbaillets et al., 2000), which are
formed by aggregation of ES cells in the presence of serum and in the absence of
leukemia inhibitory factor (LIF), a cytokine necessary for maintaining ES cells in an
undifferentiated state. EBs contain many different cell types that are fated to produce
cells of all three primary germ layers. Since there is complex intercellular signalling
between the multiple cell types of an EB, and as they are generated in the presence of
serum with its host of undefined factors, EB formation precludes a direct analysis of the
mechanisms regulating the differentiation of a specific cell lineage. In order to assess a
default state, we wanted to isolate single ES cells and minimize any exposure to extrinsic
factors that might be either instructive or inhibitory to cell fate specification. Therefore,
we employed a system of chemically-defined, serum-free, feeder layer-free culture
conditions coupled with low cell densities (to abrogate intercellular signalling). In a
previous study, we reported that these conditions appeared to favour neural determination
of ES cells (Tropepe et al., 2001). Further, a novel colony-forming primitive NSC
population arose under these conditions, one with characteristics intermediate to those of
ES cells and forebrain-derived “definitive” NSCs. Here, we demonstrate default neural
fate acquisition by ES cells, a process shown to be independent of potential instructive
factors. FGFs were found to be important for the proliferation, but not the generation, of
45
the default pathway-derived primitive NSCs. Further, we provide evidence that the
default neural fate pathway specifically gives rise to primitive NSCs, and that primitive
NSC mortality resulting from a survival challenge, which could be mitigated by survival
factors or genetic interference with apoptosis, was responsible for limiting the persistence
and proliferation of these cells.
RESULTS
ES Cells Rapidly Acquire a Default Neural Identity in Minimal Conditions
To assess the potential default fate of ES cells, we remo