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Asymmetric cell division during neurogenesis, and the mechanisms behind GABAergic cortical interneuron development and specification

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Asymmetric cell division during neurogenesis, and the mechanisms behind GABAergic cortical interneuron development and specification

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Content ASYMMETRIC CELL DIVISION DURING NEUROGENESIS, AND THE MECHANISMS
BEHIND GABAERGIC CORTICAL INTERNEURON DEVELOPMENT AND
SPECIFICATION

by

Corey Sayuri Kauai Kelsom

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

DECEMBER 2012

Copyright 2012 Corey Sayuri Kauai Kelsom

ii

Table of Contents
List of Figures iv
Abstract v
Introduction: Background on Asymmetric Division and GABAergic Interneurons 1
Asymmetric Cell Division 1
GABAergic Cortical Interneurons 3
Chapter One: Neural Progenitor Cells 5
Chapter Two: Role of Notch Signaling During Neurogenesis 6
Chapter Three: Cell Fate / Segregating Determinants 8
Numb 8
Prospero 9
Brat 10
Chapter Four: Adaptor Proteins 12
Miranda 12
Pon 13
Chapter Five: Setting Up Polarity 14
Chapter Six: Importance of Mitotic Spindle Orientation 17
Chapter Seven: Coupling Cell Cycle Regulators with Asymmetric Division Machinery 21
Chapter Eight: Consequences of Disrupting Asymmetric Cell Division 23
Chapter Nine: GABAergic Cortical Interneurons 27
Role of GABAergic Cortical Interneurons 27
Interneuronal Subtypes 27
Chapter Ten: Origin of GABAergic Interneurons 30
Medial Ganglionic Eminence 32
Caudal Ganglionic Eminence 33
Minor and Miscellaneous Sources of Cortical Interneurons 33
Embryonic Preoptic Area 33
Lateral Ganglionic Eminence 34
Rostral Migratory Stream 35
Dorsal White Matter 35
Septal Region 36

iii

Chapter Eleven: Specification of Interneurons 37
Specification of Nkx2.1-Independent Interneuron Population 40
Time Course of Interneuron Specification 41
Candidate Factors for Interneuron Specification 43
Importance of Signaling Pathways in Interneuron Specification 44
Differences in Cortical Interneuron Origin and Specification Between Rodents
and Primates 46
Clinical Implications of GABAergic Interneuron Specification 48
Chapter Twelve: Migration to the Cerebral Cortex 49
Motogens 50
Chemorepellents and Chemoattractants 51
Neurotransmitters 53
Lhx6 53
Conclusion 54
Bibliography 57

iv

List of Figures
Figure 1: Intrinsic vs. Extrinsic Modes of Asymmetric Cell Division 2
Figure 2: Some of the Key Players in Asymmetric Cell Division of Drosophila
Neuroblasts 22
Figure 3: Neuroblast Self-Renewal vs. Differentiation and Tumorigenesis 23
Figure 4: Sagittal (top) View of the Embryonic Telencephalon, Showing the
Major Origins of GABAergic Cortical Interneurons 36
Figure 5: General Schematic of GABAergic Cortical Interneuron Specification 48

v

Abstract

On the most basic level, an asymmetric division is a developmental process that produces
two daughter cells, each possessing a different identity or fate. Progenitor cells known as
neuroblasts undergo asymmetric division to produce a daughter neuroblast and another cell
known as a ganglion mother cell. There are several features of asymmetric division in
Drosophila melanogaster that make it a very complex process. The cell fate determinants that
play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical
polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs
properly. The role of mitotic spindle orientation, as well as how cell cycle regulators influence
asymmetric division machinery, will also be addressed. Most significantly, malfunctions during
asymmetric cell division have shown to be causally linked with neoplastic growth and tumor
formation. A number of neuronal types and subtypes develop upon the completion of
neurogenesis. One type of neuron that will be discussed at length is the GABAergic interneuron
of the cerebral cortex. GABAergic interneurons are inhibitory neurons of the nervous system
that are so named due to their release of the neurotransmitter gamma-aminobutyric acid
(GABA). The developmental origins of GABAergic interneurons will be discussed, as well as
factors that influence the migration routes that these interneurons must take in order to ultimately
localize in the cerebral cortex. A number of recent findings concerning the transcriptional
network of genes and candidate genes that play a role in the specification and maintenance of
GABAergic interneuron fate will be discussed. Gaining an understanding of the different aspects
of cortical interneuron development and specification, especially in humans, has useful clinical
applications that may serve to treat various neurological disorders linked to alterations in
interneuron populations.

1

Introduction: Background on Asymmetric Division and GABAergic Interneurons
Asymmetric Cell Division
Asymmetric cell division is a phenomenon that has long been studied, especially in the
developing nervous system of invertebrates and vertebrates. Asymmetric cell division is a
mechanism whereby any given cell divides to give rise to two daughter cells, each of which
possesses a different fate than the other (Horvitz and Herskowitz). Such “fates” can be
manifested as differences in size, morphology, gene expression pattern or the number of
subsequent cell divisions undergone by the two newly born daughter cells (Horvitz and
Herskowitz).
To date there are two established modes of asymmetric cell division. One type of
division, commonly referred to as a niche-controlled, or extrinsic, mechanism of cell division,
emphasizes the importance of the stem cell niche (Li and Xie). Environmental factors influence
the ability to maintain the progenitor population, and a cell relies on contact with its stem cell
niche to be able to self-renew. A second, intrinsic mechanism of asymmetric cell division serves
as the dominant mode of division during development and will be the focus of this discussion
rather than the niche-controlled mechanism. With regard to the intrinsic mechanism, regulators
of self-renewal are asymmetrically localized during mitosis, so that when cells divide only one
daughter cell inherits these regulators and thus takes on a different fate than its sister cell
(Betschinger and Knoblich; Yu, Kuo and Jan). Actively dividing Drosophila neuroblasts, which
serve as precursor and progenitor cells of the nervous system, take the intrinsic route of
asymmetric cell division. A brief background of Drosophila neural progenitor cells will be
given. Notch signaling, which is a very important component that ties into the developmental
process of neurogenesis, will also be discussed.

2

Figure 1: Intrinsic vs. Extrinsic Modes of Asymmetric Cell Division.

The major aspects of asymmetric cell division in Drosophila will be discussed at length.
An apical-basal axis of polarity is set up within cells, which is used to both asymmetrically
distribute self-renewal determinants and orient the mitotic spindle to polarize the determinants, is
a very important feature of asymmetric cell division. The cell fate determinants of neural stem
cell self-renewal and their asymmetric localization are also essential in ensuring that the
divisional machinery operates correctly. Additionally, the role that mitotic spindle orientation
plays in asymmetric division is tantamount to this developmental process and will also be
discussed. The coordination of asymmetric protein localization with cell cycle progression is
another aspect of asymmetric cell division that will be covered as well. Moreover, of great
importance to this field of research is the concept that failure of asymmetric cell division to occur
properly has widespread consequences, mainly that of neoplastic cellular growth and
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3

tumorigenesis. The developmental outcomes faced by dividing neuroblasts in Drosophila when
asymmetric cell division machinery is altered or lost will be elaborated upon. In particular, the
repercussions of disruption of cortical polarity due to missegregated cell fate determinants and
proteins, as well as misalignment of the mitotic spindle, will be discussed. Existence of this
undeniable link between asymmetric cell division gone awry and tumorigenesis shows that
understanding the mechanisms behind asymmetric cell division hold great value not only on a
developmental basis, but on the clinical level as well.

GABAergic Cortical Interneurons
Interneurons play a vital role in the wiring and circuitry of the developing nervous system
of all organisms, both invertebrates and vertebrates alike. Generally speaking, an interneuron is
a specialized type of neuron whose primary role is to form a connection between other types of
neurons. They are neither motor neurons nor sensory neurons, and also differ from projection
neurons in that projection neurons send their signals to more distant locations such as the brain or
the spinal cord. Of great importance is that interneurons function to modulate neural circuitry
and circuit activity (Hensch; Owens and Kriegstein; X. J. Wang et al.; Whittington and Traub).
Interneurons of the central nervous system are of the inhibitory type. In contrast to excitatory
neurons, inhibitory cortical interneurons characteristically release the neurotransmitters gamma-
aminobutyric acid (GABA) and glycine (Dreifuss, Kelly and Krnjevic; Fonnum and Storm-
Mathisen; Somogyi, Freund, et al.). Cortical interneurons are so named for their localization in
the cerebral cortex, which is defined as a sheet of outer neural tissue, which functions to cover
the cerebrum and cerebellum structures in the brain. An emphasis will be placed upon on the
function and origin of GABAergic cortical interneurons of the developing nervous system.

4

Within the overarching categorization of GABAergic interneurons there are also numerous
interneuron subtypes, which will be elaborated upon in this discussion as well. The migratory
route that they take, as well as factors that influence their specification as interneurons, will also
be addressed. Most recently, there has been a move to understand the transcription factors that
influence cortical interneuron specification and subtype. The most arduous task that researchers
face is understanding the mechanism by which each interneuron subtype is specified as well as
the various genes and transcription factors that may be involved; this is not helped by the fact
that there are so many subtypes whose features often overlap with one another. The most recent
findings concerning construction of a genetic/transcriptional network will be discussed at length.

5

Chapter One: Neural Progenitor Cells
Neuroblasts serve as the progenitor cell population in the developing Drosophila nervous
system, and demonstrate the importance of asymmetric cell division in generating terminally
differentiated neurons and glia. There are two types of neuroblasts in the developing nervous
system – embryonic neuroblasts, which give rise to the simple nervous system present in larva,
and larval neuroblasts, which generate the neurons in the fly’s adult nervous system.
Neuroblasts initially divide symmetrically – one neural progenitor cell divides to produce
two identical, daughter neuroblasts, thereby maintaining and expanding the population of neural
stem cells. As neural development progresses, neuroblasts then undergo asymmetric cell
divisions. During division, one daughter cell is produced that is identical to its parent, and
therefore maintains a neuroblast identity. The second daughter that is generated is smaller in size
and is referred to as a ganglion mother cell (GMC). Ganglion mother cells proceed to undergo
one last division to ultimately generate two differentiating neurons.
Miyata and colleagues have recently investigated the mechanisms in place that are known
to regulate the number of neurons during neural development in the mouse. In short, the
duration of neurogenesis, the rate of neural progenitor cell division, and the mode of neural
progenitor division are the key determining factors of the total number of neurons generated
during neocortical development (Miyata et al.). “Mode of neural progenitor division” is defined
as the generation of: (1) two neural progenitors, (2) one neural progenitor and one neuron, or (3)
two neuronally committed cells (Miyata et al.).

6

Chapter Two: Role of Notch Signaling During Neurogenesis
The Notch signaling pathway has been shown to function as a key regulator in the
developing nervous system. Findings from numerous studies contribute to the fact that Notch
signaling controls the balance between self-renewal and differentiation of neural progenitor cells
(Fortini; Kageyama et al.; Kopan and Ilagan), and elegantly coordinates neuroblastic asymmetric
cell division. Neuronal differentiation is triggered by transcriptional activators such as Mash1
and Neurogenin2 (Ngn2) (Bertrand, Castro and Guillemot; Ross, Greenberg and Stiles). Mash1
and Ngn2 simultaneously activate the expression of the Notch receptor ligands Delta1, which
activates Notch in neighboring cells. Neuralized (Neur) is an E3 ubiquitin ligase protein that
facilitates the endocytosis of Delta and the extracellular domain of Notch (Lai et al.; Lai and
Rubin "Neuralized Is Essential for a Subset of Notch Pathway-Dependent Cell Fate Decisions
During Drosophila Eye Development"; Lai and Rubin "Neuralized Functions Cell-
Autonomously to Regulate a Subset of Notch-Dependent Processes During Adult Drosophila
Development"; Pavlopoulos et al.; Yeh et al.). Notch activation is then followed by nuclear
transport of the Notch intracellular domain (NICD) and subsequent formation of a transcription
activator complex (Honjo). This complex triggers the activation of Hes1 and Hes5, which act to
repress the expression of proneural genes in neighboring progenitor cells (Kageyama, Ohtsuka
and Kobayashi). This achieves the purpose of asymmetric cell division: it ensures the formation
of differentiating neurons while simultaneously allowing neighboring cells to remain as neural
progenitors.
Updates to the study of Notch signaling and its role in asymmetric cell division of
neuroblasts have recently been made. Monastirioti and colleagues have identified Drosophila
Hey as a target of Notch during neurogenesis (Monastirioti et al.). Hey is a basic-helix-loop-

7

helix-Orange (bHLH-O) transcription factor that is expressed primarily in the population of
neuroblasts possessing activated Notch signaling, and therefore is thought to contribute to
maintaining/expanding the neural progenitor cell population during development (Monastirioti et
al.). A recent study has also investigated other candidate genes that Notch signaling regulates in
the context of neurogenesis. A gene referred to as Deadpan (Dpn) that encodes a bHLH
transcription factor is another direct target of the Notch signaling pathway; so far this has only
been demonstrated in intermediate progenitor cells (San-Juan and Baonza). Overexpression of
Dpn in INPs gives rise to a cancer-like phenotype in which neuroblasts overproliferate
inappropriately (San-Juan and Baonza).
The first study to show the link between both asymmetric division machinery and Notch
signaling and specification of a neurotransmitter neuronal phenotype was performed in
Drosophila by Tio et al (Tio et al.). Findings from this study showed that loss of function of the
proteins Inscuteable and Bazooka resulted in an excess number of dopaminergic neurons. On the
other hand, loss of function of basally distributed proteins such as Numb resulted in a reduction
of dopaminergic neurons (Tio et al.). Additionally, loss of Notch signaling results in an excess
amount of this neuronal phenotype, and vice versa. Given the high levels of conservation
between Drosophila and higher-level organisms, this study may hold great promise in
understanding the link between asymmetric division and neuronal specification.
Notch signaling regulates a vast amount of developmental processes in Drosophila,
including that of optic lobe development (W. Wang et al.). Briefly, loss of function analyses of
both Notch and Delta demonstrate that Notch signaling might play a dual role here: maintenance
of neuroepithelial stem cell population and inhibition of these stem cells toward differentiation
into medulla neuroblasts (W. Wang et al.).

8

Chapter Three: Cell Fate / Segregating Determinants
Segregating determinants, also referred to as cell-fate determinants, are proteins that play
a crucial role in specifying daughter cell fate. It is the asymmetric localization of these particular
determinants (in addition to other factors) to the basal side of the dividing neural progenitor cell
that is largely thought to produce two daughter cells, each with a different fate.

Numb
Numb is a transcription factor that was originally identified in Drosophila sensory organ
precursor (SOP) cells and has been demonstrated to segregate asymmetrically in neuroblasts
(Rhyu, Jan and Jan). Numb has been shown to serve as a tissue-specific repressor of the Notch
signaling pathway (Le Borgne, Bardin and Schweisguth; Schweisguth); it binds alpha-adaptin
and potentially plays a role in directing intracellular transport of Notch intermediates (Berdnik et
al.). Loss or disruption of Numb function in the larval brain manifests in the overproliferation of
mutant neuroblasts, which therefore gives rise to a tumor-like phenotype (C. Y. Lee, R. O.
Andersen, et al.; H. Wang, G. W. Somers, et al.).
More recently, the role of additional proteins in regulating the asymmetric localization of
Numb has been investigated. Wang and colleagues demonstrated that protein phosphatase 2A
(PP2A) is a brain tumor suppressor protein that forms a heterotrimeric complex that functions to
inhibit the self-renewal of neuroblasts (C. Wang et al.). The PP2A complex regulates, among
other things, the asymmetric localization and phosphorylation of Numb (C. Wang et al.).
Additionally, the Hem/Kette/Nap1 protein has been shown to play a very important role in the
asymmetric division of Drosophila neuroblasts; it does so by regulating the localization of Numb
and another adaptor protein known as Inscuteable (Zhu and Bhat). Hence, Hem/Kette/Nap1

9

mutant GMCs display symmetric, rather than asymmetric division. Neur, a protein previously
established to play a role in Notch signaling, has recently been shown to promote the asymmetric
localization of Numb by downregulating expression of the transcription factor Pdm1 (Bhat,
Gaziova and Katipalla). This function is evidenced by mutational analaysis, which shows that
Numb is symmetrically (rather than asymmetrically) localized in Neur mutants. Moreover, Neur
overexpression results in expansion of the neuroblast population at the expense of differentiating
neurons (Bhat, Gaziova and Katipalla).

Prospero
A second cell-fate determinant that has also been shown to segregate asymmetrically in
neuroblasts is the transcription factor Prospero. Choksi and colleagues have demonstrated on the
genome-wide level that Prospero (abbreviated Pros) has several hundred binding sites in the
Drosophila genome (Choksi et al.). Importantly, this study showed that Pros acts as a “switch”
between neuroblast self-renewal and differentiation: it has the ability to repress neuroblast and
cell-cycle genes, as well as regulate neural differentiation genes (Choksi et al.).
Pros mutant GMCs fail to commit to a differentiated, neuronal fate: these mutant cells
have prolonged expression of neuroblast markers and inappropriately continue to divide (Choksi
et al.). It had previously been postulated that the upregulation of cell cycle regulators, mainly
Cyclin A, Cyclin E, and Cdc25, may be the reason for this occurrence in Pros mutant neuroblasts
(Li and Vaessin). More recently, Berger et al have demonstrated that Cyclin E possesses cell-
cycle independent roles in asymmetric division: it inhibits Pros function, and may also serve to
regulate the cortical localization of Pros, hence allowing neuroblasts to maintain their identity
rather than committing to the neuronal lineage (C. Berger et al.). By regulating Pros localization,

10

CycE therefore plays a crucial role in maintaining the neural progenitor population. Similar to
Numb, mutational analysis shows that Pros mutations give rise to stem cell-derived tumors in
larval neuroblasts (Bello, Reichert and Hirth; Betschinger, Mechtler and Knoblich "Asymmetric
Segregation of the Tumor Suppressor Brat Regulates Self-Renewal in Drosophila Neural Stem
Cells"; C. Y. Lee, B. D. Wilkinson, et al.).
In addition to its role as a cell fate determinant in GMCs, Pros also possesses a role in
coupling cell cycle progression to neurogenesis during development: its transient expression
ensures that neuronally-committed cells exit the cell cycle at the appropriate time (Colonques et
al.).

Brat
Brat is the third cell-fate determinant and growth inhibitor that was discovered to play a
role in regulating the balance between neuroblast self-renewal and differentiation (Bello,
Reichert and Hirth; Betschinger, Mechtler and Knoblich "Asymmetric Segregation of the Tumor
Suppressor Brat Regulates Self-Renewal in Drosophila Neural Stem Cells"; C. Y. Lee, B. D.
Wilkinson, et al.). During neural development, Brat (in combination with Pros) segregates
asymmetrically into only one of two daughter neuroblasts to specify GMC fate (Betschinger,
Mechtler and Knoblich "Asymmetric Segregation of the Tumor Suppressor Brat Regulates Self-
Renewal in Drosophila Neural Stem Cells"). Similar to Numb, loss of Brat results in both
daughter cells taking on a neuroblast identity, which ultimately gives rise to a tumor phenotype
(Betschinger, Mechtler and Knoblich "Asymmetric Segregation of the Tumor Suppressor Brat
Regulates Self-Renewal in Drosophila Neural Stem Cells"). Further mutational analyses have
demonstrated that the phenotype of Pros/Brat double mutants is one that lacks most, if not all,

11

GMCs due to an overexpansion of neuroblasts (Betschinger, Mechtler and Knoblich
"Asymmetric Segregation of the Tumor Suppressor Brat Regulates Self-Renewal in Drosophila
Neural Stem Cells"). Observations of these mutant phenotypes have led to the speculation that
Brat may function to inhibit cell growth in one of two newly born neuroblast daughters so as to
generate one neuron and one neuroblast, rather than two neuroblasts (Knoblich). The true
molecular mechanism by which Brat operates, however, remains to be clarified.

12

Chapter Four: Adaptor Proteins
While proper function of the segregating determinants is crucial for asymmetric cell
division, adaptor proteins are just as important in ensuring that division is properly executed.
Adaptor proteins facilitate the asymmetric localization of Numb, Pros and Brat.

Miranda
Miranda serves as the adaptor protein for segregating determinants Pros, Brat, and
Staufen, although Staufen’s functions will not be covered. For more details regarding Staufen,
refer to Betschinger and Knoblich (Betschinger and Knoblich). Miranda behaves similarly to
Pros and Brat; in dividing neuroblasts it too localizes asymmetrically and segregates into one of
the two daughter cells (Betschinger and Knoblich). Importantly, when Miranda is mutated, both
Pros and Brat segregate symmetrically rather than asymmetrically in dividing neuroblasts, and
the cell-fate determinants are therefore uniformly cytoplasmic.
Atwood and colleagues have more recently shown that atypical protein kinase (aPKC),
which is a regulator of cell polarity, directly phosphorylates Miranda (Atwood and Prehoda).
Phosphorylation displaces Miranda from the apical cortex, where it can then work to polarize the
cell-date determinants. These findings counter the theory that Miranda is regulated by a more
complicated cascade involving aPKC, Lgl and myosin II (Atwood and Prehoda). Regardless of
the mechanism by which it works, Miranda is a crucial adaptor protein that connects Pros and
Brat to the machinery for asymmetric protein localization.

13

Pon
Pon, which stands for Partner of Numb, is so-named because of its function: it is the
adaptor protein for Numb and therefore binds to Numb. Unlike Miranda, however, Pon is not
required for the asymmetric localization of Numb (H. Wang, Y. Ouyang, et al.). In the absence
of Pon, localization of Numb is delayed in metaphase, which therefore results in a defect in the
neuroblast self-renewal (H. Wang, Y. Ouyang, et al.).

14

Chapter Five: Setting Up Polarity
While the asymmetric localization of cell-fate determinants and the functions they play in
neuroblast self-renewal or neuronal commitment has been established, the question of how they
are directed to the basal cortex remains to be answered. The answer lies within an axis of
polarity that is set up during interphase. Cell-fate determinants and the orientation of stem cell
division both take instruction from this axis of polarity, which consists of aPKC and the Par
proteins Par-3 (Bazooka in Drosophila) and Par-6 (Goldstein and Macara; Suzuki and Ohno).
Par-3, Par-6 and aPKC are required for establishing apical-basal polarity in developing
neuroblasts: they concentrate to the apical cell cortex of the neuroblast (Knoblich). The
localization of these proteins is opposite of the location that the cell-fate determinants
concentrate in mitosis, and their presence ensures that the determinants are segregated into the
basal cell cortex. When any one of the three polarity proteins is mutated, the cell-fate
determinants are distributed uniformly in the cell cortex, and mitotic spindles orient randomly.
Hence, aPKC and the Par proteins are critical for constructing a “blue-print”, in which the cell-
fate determinants are properly distributed and the mitotic spindle is properly oriented.
Due to the extensive amount of literature in existence concerning the role of the Par
proteins, this discussion will mainly emphasize the role that Lgl, the substrate of aPKC, plays in
the asymmetric localization of segregating determinants.
Lethal (2) giant larvae (abbreviated Lgl) was identified by several groups as the key
substrate for aPKC (Betschinger, Mechtler and Knoblich "The Par Complex Directs Asymmetric
Cell Division by Phosphorylating the Cytoskeletal Protein Lgl"; Plant et al.; Yamanaka et al.).
Unlike the other polarity proteins that are apically localized, Lgl is uniformly distributed
throughout the cortex. Lgl is necessary for ensuring that the cell-fate determinants are brought to

15

the cell cortex and localized asymmetrically during mitosis (Ohshiro et al.; Peng et al.). The
mechanism by which Lgl is able to achieve its purpose was elucidated soon after, and concerns
the Par proteins, mainly aPKC. aPKC is responsible for phosphorylating Lgl on three conserved
serines at the cell cortex (Betschinger, Mechtler and Knoblich "The Par Complex Directs
Asymmetric Cell Division by Phosphorylating the Cytoskeletal Protein Lgl"). This
phosphorylation event is proposed to prevent Lgl from associating with the actin skeleton, and
most significantly, prevents the cell fate determinants from localizing apically (Betschinger,
Mechtler and Knoblich "The Par Complex Directs Asymmetric Cell Division by Phosphorylating
the Cytoskeletal Protein Lgl"). aPKC-mediated phosphorylation of Lgl seems to somehow
inactivate Lgl, and is evidenced by the phenotype of aPKC overexpression, which resembles that
of Lgl loss of function.
Adding more complexity to the puzzle is the fact that aPKC also possesses the ability to
phosphorylate segregating determinants directly, rather than acting on Lgl first. Smith and
colleagues have demonstrated that aPKC can directly phosphorylate Numb, whereby Numb is
transported from the cell cortex into the cytoplasm (Smith et al.).
In a 2008 review, Knoblich has proposed a model to account for aPKC and Lgl activity in
neuroblasts, whereby aPKC, which is localized at the apical cortex, functions to restrict Lgl to
the basal side of the neuroblast (Knoblich). Given Lgl’s responsibility of recruiting the cell-fate
determinants to the cortex, this makes sense, since the determinants only localize basally. While
this model is certainly logical, the molecular mechanism by which Lgl operates has not yet been
elucidated.
Recent studies have delved into mechanisms underlying aPKC function. Chang et al, for
instance, have shown that Zif is a transcription factor and zinc finger protein that is required for

16

aPKC to both be expressed and asymmetrically localized (Chang et al.). Zif acts to directly
repress transcription of aPKC, and in turn, aPKC phosphorylates Zif, which ultimately leads to
Zif inactivation in neuroblasts. The combined actions of these two proteins thus play an
indispensable role in setting up cortical polarity and also controlling progenitor self-renewal
(Chang et al.).

17

Chapter Six: Importance of Mitotic Spindle Orientation
The significance of mitotic spindle orientation in regulating neuroblast division has also
been extensively studied and renewed (Morin and Bellaiche; Siller and Doe "Spindle Orientation
During Asymmetric Cell Division"). Here, the coordination between mitotic spindle orientation
and asymmetric localization of the segregating determinants will be discussed.
Kraut and colleagues first determined that a protein known as Inscuteable (Insc) plays an
immensely important role in coordinating mitotic spindle alignment and localization of the cell-
fate determinants to the basal cortex in dividing neuroblasts (Kraut et al.). Inscuteable operates
by binding the polarity protein Bazooka (Par-3) in the apical region of neuroblasts, and recruits
another protein called Pins (which will be discussed below) (Nipper et al.). Zhu and Bhat have
recently shown that the Drosophila protein Hem/Kette/Nap1 also regulates localization of
Inscuteable. As was previously discussed, this protein regulates the asymmetric division of
neural progenitors by controlling Numb localization (Zhu and Bhat).
It is important to note that the binding of Insc to Pins triggers the activation of two
downstream pathways that participate in mitotic spindle positioning, both of which are mediated
by Pins. The first pathway is often referred to as the Pins-Mud pathway. Pins is a protein whose
structural features are functionally purposeful: it contains three domains called GoLoco domains
in its C-terminal region; these domains bind Gαi, which is a heterotrimeric G protein subunit
(Nipper et al.). Upon binding, Pins is recruited to the plasma membrane and switches from and
switches from an inactive to active state. It is in this active state that the N-terminal region of
Pins binds to another protein called Mud (which stands for Mushroom body defect) (Bowman et
al.; Izumi et al.; Siller, Cabernard and Doe). Mud is the Drosophila homolog of NuMA, and is
thought to play a role in recruiting the Dynein/Dynactin complex (Siller and Doe "Lis1/Dynactin

18

Regulates Metaphase Spindle Orientation in Drosophila Neuroblasts"). This complex functions
to generate pulling forces on astral microtubules so as to further advance mitotic spindle
positioning (Siller and Doe "Lis1/Dynactin Regulates Metaphase Spindle Orientation in
Drosophila Neuroblasts").
The second pathway that is activated upon Insc binding Pins is the Dlg pathway
(Johnston et al.; Siegrist and Doe). In a 2005 study, Siegrist and Doe elegantly showed that
astral microtubules bind to a kinesin referred to as Khc-73 as well as the protein Discs large
(Dlg). Of note is that the Dlg pathway functions during metaphase to coordinate neuroblast
polarity with the mitotic spindle, independent from the Pins-Mud pathway (Siegrist and Doe).
Recently, others have further investigated the mechanisms by which Inscuteable exerts its
effects on mitotic spindle positioning through Pins. Results from a study by Mauser and Prehoda
have suggested that Insc preferentially inhibits the Mud pathway, while enabling continued
activation of the Dlg pathway (Mauser and Prehoda). A variety of rationales may explain these
findings, one of them being assurance that the spindle is attached to the cortex via Dlg before
spindle pulling forces are activated via the Mud pathway (Mauser and Prehoda).
A 2011 study has characterized the Drosophila cytoplasmic polyadenylation element
binding (CPEB) protein Orb2. CPEB proteins function to bind mRNAs in order to control their
localization and subsequent translation. Hafer and colleagues report that Orb2 functions in the
asymmetric division of both stem and precursor cells in the context of the developing Drosophila
nervous system (Hafer et al.). Additionally, Orb2 mutants present with disrupted mitotic spindle
alignment; results from this study suggest that it may serve to promote the localization of aPKC
(Hafer et al.).

19

Speicher et al were first to demonstrate that the PDZ protein Canoe (Cno) plays a role in
both the localization of cell-fate determinants and orientation of the mitotic spindle in
asymmetrically dividing neuroblasts (Speicher et al.). Cno apically localizes with the
Bazooka/Par-3 in neuroblasts, and was also found to be essential for proper distribution of cell-
fate determinants on the basal side of the cell; importantly, failure of the determinants to basally
distribute resulted in misorientation of the mitotic spindle. This study further demonstrated that
Cno interacts with the proteins Inscuteable, Gαi, and Mud, and acts downstream of apical
proteins Insc-Pins-Gαi, but upstream of Mud (Speicher et al.). More recently confirming Cno’s
involvement in regulating mitotic spindle orientation and neuroblast cortical polarity is a study
demonstrating that Rap1, a Ras-like small guanosine triphosphatase, signals through Cno and
another guanine nucleotide exchange factor known as Rgl in order to regulate neuroblast polarity
(Carmena, Makarova and Speicher). Carmena and colleagues postulate that Rap1 forms a novel
Rap1-Rgl-Ral signaling network that interacts with other apical proteins to influence neuroblast
cortical polarity and spindle orientation: loss of function of Rap1, Rgl and Ral proteins affect
both spindle orientation and the localization of proteins Mud and Cno (Carmena, Makarova and
Speicher).
Most recently, a protein known as Huntingtin (abbreviated Htt) has been shown to
regulate mitotic spindle orientation in Drosophila neuroblasts as well as in mammalian cortical
progenitor cells (Godin et al.). Htt is mutated in Huntington’s disease, a neurodegenerative
disorder caused by a genetic defect on chromosome 4. Godin et al showed that RNAi-mediated
knockdown of Htt prevents proper spindle orientation; this occurs due to the incorrect
localization of the p150
Glued
subunit of dynactin, dynein, and the NuMA protein (Godin et al.).

20

In addition to demonstrating its role in controlling mitosis, further elucidation of Htt’s role may
hold great promise in the therapeutic treatment of Huntington’s disease.

21

Chapter Seven: Coupling Cell Cycle Regulators With Asymmetric Division Machinery
While the proper localization of asymmetrically segregating proteins and cell fate
determinants is of utmost importance to ensure that neuroblasts divide normally, cell cycle
regulators also play a role in controlling the asymmetric division machinery. Two such cell
cycle regulators are Aurora-A (abbreviated Aur-A) and Polo, which are serine/threonine kinases.
Aur-A has been shown to inhibit the self-renewal of Drosophila larval neuroblasts and promote
neuronal differentiation (C. Y. Lee, R. O. Andersen, et al.). Mutational analyses demonstrate
that Aur-A mutant neuroblasts undergo unrestricted self-renewal, and this phenotype is due to
both abnormal aPKC/Numb cortical polarity and misalignment of the mitotic spindle (C. Y. Lee,
R. O. Andersen, et al.). Another study corroborates these findings: an excessive number of
neuroblasts is observed in Aur-A mutants, thereby showing that Aur-A acts as a tumor
suppressor (H. Wang, G. W. Somers, et al.). A later study revealed the molecular mechanism for
the asymmetric localization of Numb: Aur-A phosphorylates Par-6, which in turn activates aPKC
and phosphorylates Lgl; this even allows Bazooka to enter the complex, thereby allowing aPKC
to regulate Numb localization to one side of the cell cortex (Wirtz-Peitz, Nishimura and
Knoblich). Polo is another cell cycle regulator that has a hand in asymmetric cell division.
Like Aur-A, Polo also acts as a tumor suppressor: excessive neuroblast numbers are observed in
Polo mutants (H. Wang, Y. Ouyang, et al.). Wang and colleagues have demonstrated that Polo
phosphorylates Partner of Numb (Pon), and this is important for Pon to be able to localize Numb
(H. Wang, Y. Ouyang, et al.).
What must also be taken into account are the phosphatases that counteract the effects of
the aforementioned kinases. Protein phosphatase 4A (PP4A) and protein phosphatase 2A
(PP2A) also play a role in the division of Drosophila neuroblasts. The mechanisms by which

22

these phosphatases operate to regulate are more complicated (Chabu and Doe; Krahn, Egger-
Adam and Wodarz; Ogawa et al.) and will not be discussed here; for more details refer to the
review by Chang et al (Chang, Wang and Wang). Although progress has been made in terms of
understanding how cell cycle is coupled to asymmetric division machinery, a more widespread
and thorough analysis should be performed to identify candidate cell cycle genes involved
asymmetric cell division.

Figure 2: Some of the Key Players in Asymmetric Cell Division of Drosophila Neuroblasts.

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23

Chapter Eight: Consequences of Disrupting Asymmetric Cell Division
It has long been postulated that tumorigenesis and uncontrolled cellular replication may
be causally linked to asymmetric cell division gone awry. Several studies have utilized
Drosphila transplantation models to investigate this concept, one in particular being a
Drosophila transplantation model of neural stem cell-derived cancer (Laurenson et al.). RNAi-
mediated knockdown of cell fate determinants Numb, Brat, and Prospero in neuroblasts resulted
in neoplastic tumor formation after transplantation (Laurenson et al.). Transplantation models
are clearly helpful in gaining an understanding of what happens when asymmetric divisional
machinery fails to work properly in order to better understand neoplastic growth and tumor
formation.

Figure 3: Neuroblast Self-Renewal vs. Differentiation and Tumorigenesis.

It is evident that the repercussions of failure of neuroblasts to divide asymmetrically are
dire. It also follows that proteins whose functions prevent proper execution of asymmetric cell
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24

division rather than allow inappropriate overproliferation of neuroblasts may be viewed as tumor
suppressors (Ohshiro et al.; Peng et al.; C. Wang et al.). Several transplantation experiments, as
well as studies that have been discussed thus far, unequivocally show the correlation between
asymmetric cell division machinery and cancer- or tumor-like phenotypes in the context of the
nervous system (Caussinus and Gonzalez).
The missegregation of apically- and/or basally localized proteins is a major causal factor
for neoplastic growth and tumorigenesis in neuroblasts. Take, for instance, brain tissue
possessing neuroblasts with mutated versions of Pins, Miranda, Numb, or Prospero, all crucial
asymmetric cell division proteins: transplantation experiments with such brain tissue invariably
results in inappropriate neuroblast overproliferation and gives rise to a cancer-like phenotype and
ultimately, death (Zhong and Chia). Additionally, a plethora of studies have demonstrated that
absence or disruption of proper cell-fate determinant function (Numb, Pros, Brat) results in an
uncontrollable expansion of the neuroblast/progenitor pool, and heavy (or complete) depletion of
neuronally committed cells (Bello, Reichert and Hirth; Betschinger, Mechtler and Knoblich
"Asymmetric Segregation of the Tumor Suppressor Brat Regulates Self-Renewal in Drosophila
Neural Stem Cells"; Choksi et al.; C. Y. Lee, R. O. Andersen, et al.; C. Y. Lee, B. D. Wilkinson,
et al.; H. Wang, Y. Ouyang, et al.; H. Wang, G. W. Somers, et al.). Simultaneous disruption of
both Pins and Lgl proteins results in unrestricted growth of neuroblasts due to the delocalization
of aPKC (Lee, Robinson and Doe). Additionally, results from this same study demonstrated that
overexpression of a membrane-targeted form of aPKC results in a significant increase in number
of neuroblasts (Lee, Robinson and Doe). The opposite is also true: loss or reduction of aPKC
expression results in a corresponding reduction of neuroblast numbers (Lee, Robinson and Doe;
Rolls et al.).

25

Because aPKC clearly serves an important role in maintaining a balance between
neuroblast self-renewal and differentiation, it is logical to determine which molecules or factors
regulate aPKC itself. Two previously mentioned proteins, Protein phosphatase PP2A and the
zinc finger protein known as Zif, have been shown to negatively regulate aPKC (Chang et al.; C.
Wang et al.). Zif binds to a region of aPKC, thereby repressing aPKC transcription (Chang et
al.). There is, however, a more complicated feedback mechanism between aPKC and Zif that
has yet to be completely elucidated in the context of regulation of neuroblast self-renewal. As
these studies have shown, it is therefore of utmost importance that these determinants (and their
adaptor proteins) segregate properly so as to maintain the pool of GMCs that eventually give rise
to the differentiating neurons of the developing nervous system.
Just as the loss of segregating determinants leads to overgrowth and tumor formation, so
too will the loss of machinery controlling mitotic spindle orientation. Several studies have
demonstrated that improper mitotic spindle orientation causes improper segregation of proteins
that are normally asymmetrically localized. The improper segregation of said proteins in turn
leads to unrestricted neuroblast division and outgrowth. As was previously mentioned, loss of
Mud, a protein crucial for orienting the mitotic spindle, results in abnormal proliferation of
neuroblasts (Bowman et al.; Izumi et al.; Siller, Cabernard and Doe). Additionally, neuroblasts
possessing double mutations for Lgl and Pins, as well as Dlg/Gβγ double mutants (Gβγ is a
cortically localized protein that also regulates spindle orientation), demonstrate significant
overproliferation relative to wild-type (Kitajima et al.; Lee, Robinson and Doe).
Additionally, in a 2009 study, Cabernard and Doe attempted spindle disruption without
altering cell polarity via live imaging of polarity markers and spindle orientation over a time
period of multiple divisions, then analyzed cell fate utilizing molecular markers (Cabernard and

26

Doe). Results from this study suggest that when the spindle is oriented orthogonally to apical-
basal polarity, the cell-fate determinants fail to localize symmetrically rather than
asymmetrically, and both daughter cells ultimately form neuroblasts (Cabernard and Doe).
Maintaining mitotic spindle orientation is thus crucial for maintaining the neuroblast population,
ensuring that differentiating neurons are formed and preventing unwanted tumor formation.

27

Chapter Nine: GABAergic Cortical Interneurons
Role of GABAergic Cortical Interneurons
Given that the population of GABAergic interneurons in the brain is such a
heterogeneous one, it is only logical that the many different classes of interneurons will have a
myriad of roles to play in the adult nervous system. GABAergic neurons play an inhibitory role
and synaptically release the neurotransmitter GABA in order to regulate the firing rate of target
neurons. Neurotransmitter release typically acts through postsynaptic GABA
A
ionotropic
receptors in order to trigger a neuronal signaling pathway.
This research field typically organizes interneuron role/function into three components:
(1) afferent input, (2) intrinsic properties of the interneuron, and (3) targets of the interneuron.
Generally speaking, interneurons receive input from various sources, including pyramidal cells
as well as cells from other cortical and subcortical regions (Gibson, Beierlein and Connors;
Porter, Johnson and Agmon). With regard to output, cortical interneurons engage in feed-
forward and feedback inhibition (T. K. Berger et al.; Silberberg, Gupta and Markram; Y. Wang,
M. Toledo-Rodriguez, et al.). Regardless of the mode of output, the cortical interneuronal
network is further complicated by the fact that a single cortical interneuron is capable of making
multiple connections with its excitatory neuronal target(s) (Somogyi, Tamas, et al.). The
following section will elaborate upon the various subtypes of interneurons and their individual
functions.

Interneuronal Subtypes
Interneuron categorization is based on a number of features such as morphology,
immunohistochemical profile, firing pattern, axonal targeting, and electrophysiology (Ascoli et

28

al.; Cauli et al.; DeFelipe; Gupta, Wang and Markram; Kawaguchi and Kondo), it is estimated
that there are over 20 different subtypes of GABAergic interneurons in the cortex (Klausberger
and Somogyi; Parra, Gulyas and Miles). Determining which categorizing parameters to use,
however, is a difficult task, since the overall population of GABAergic progenitors is very
heterogeneous and many subtypes may overlap in certain characteristics. In the recent years
there has been a push by researchers to create a consistent nomenclature for the varying
interneuronal subtypes; a 2005 conference in Petilla, Spain, was held to accomplish this task
(Ascoli et al.). Among the numerous cortical interneuronal subtypes that exist in the cortex, the
four primary populations of GABAergic interneurons that are organized on the basis of the
individual markers they express will be discussed.
Parvalbumin- (PV) expressing interneurons are known to represent the largest subtype of
cortical interneurons; they make up approximately 50% of all GABAergic interneurons (Vitalis
and Rossier). PV-positive interneurons are most commonly referred to as the “fast-spiking”
subtype; this class of neurons is derived from the ventral medial ganglionic eminence (discussed
below). The fast-spiking subtype may take on a large basket, nest basket, or chandelier
morphology, depending on the nature of their targets. The second class of characterized
interneurons is referred to as somatostatin- (SST) expressing interneurons, which account for
approximately 30% of GABAergic interneurons. SST-positive neurons are known as Martinotti
cells, target both proximal and distal dendrites, and originate in the ventral region of the medial
ganglionic eminence (Karube, Kubota and Kawaguchi). These cells are preferentially located in
the deeper cortical layers (Markram et al.; Somogyi, Tamas, et al.). A third class of interneurons
is termed vasoactive intestinal peptide (VIP)-bipolar interneurons, which represent roughly 15%
of the GABAergic population. These neurons possess a small bipolar or double bouquet

29

morphology and primarily target interneurons. Lastly, Neuropeptide Y- (NPY) expressing
interneurons comprise a more recently distinguished group of interneurons. They make up a
relatively smaller percentage of GABAergic interneurons, and function in the slow GABAergic
inhibition of pyramidal cells and other interneurons (Olah et al.). In addition to strongly
expressing NPY, this subtype possesses neurogliaform morphology and primarily occupies
superficially located cortical layers. While these interneuronal subclasses encompass the
majority of GABAergic interneurons, it is also important to remember that there are many more,
relatively minor subtypes that have not been discussed. Additionally, there are other ways to
categorize and classify these interneurons, as many characteristics may overlap given that this
population is so heterogeneous.

30

Chapter Ten: Origin of GABAergic Interneurons
The various origins of GABAergic interneurons have been extensively studied. The
medial ganglionic eminence and the caudal ganglionic eminence are the two major sources of
GABAergic interneurons. Studies documenting other potential and relatively minor sources of
cortical interneurons will be touched upon as well.
Throughout embryogenesis, interneurons are primarily generated in a structure broadly
termed the ganglionic eminence (Corbin and Butt). The ganglionic eminence is defined as a
transitory brain structure located in the ventral area of the telencephalon, and is anatomically
present during embryonic development. The ganglionic eminence becomes evident at
approximately E11.5 in the developing murine system; this corresponds to roughly 5-6 weeks
gestation in humans. As embryonic development continues, the GEs grow and ultimately fuse,
at which point they are no longer present in the mature brain. In total there are three ganglionic
eminences: the medial ganglionic eminence (MGE), the caudal ganglionic eminence (CGE), and
the lateral ganglionic eminence (LGE). The names of the different areas within the GE are based
on their rostral-caudal location in the telencephalon. The MGE and CGE are the primary sources
of cortical interneurons in the developing nervous system.
Several crucial studies during the 1980s demonstrated that contrary to the previous
postulation that cortical interneurons originate in the underlying cortical ventricular zone, the
majority of GABAergic interneurons are actually born in the ganglionic eminences. Van Eden
and colleagues first showed, utilizing analysis of GABA-positive cells in the developing rodent
cerebral cortex, that cells presumed to be GABAergic interneurons seemed to be migrating away
from the subpallium (Van Eden et al.). This crucial observation spawned several other studies
that subsequently discovered that indeed, interneurons must migrate from the subpallium to their

31

final destinations in the cortex. To corroborate Van Eden et al’s findings, BrdU labeling of cells
in the subpallium were found to accumulate in the cerebral cortex throughout the course of
neurogenesis; importantly, these BrdU labeled cells were also GABA-positive, implicating these
cells as cortical interneurons (DeDiego, Smith-Fernandez and Fairen). Additional studies
utilizing fluorescent dye labeling of neurons and an in vitro migratory assay with DiI showed
that these interneurons display the same migratory pattern from the ganglionic eminence to the
mature cerebral cortex (de Carlos, Lopez-Mascaraque and Valverde; Tamamaki, Fujimori and
Takauji). Use of retroviruses to track neural cell lineage also contributed the early yet important
study that excitatory and inhibitory neurons are actually generated independently from one
another and therefore do not share a common lineage (Mione et al.; Parnavelas et al.). Rakic and
Lombroso later confirmed that the telencephalon consists of two separate domains: one
containing the GEs that serves as the source of inhibitory, GABAergic interneurons, and a
second in which excitatory neurons are generated (Rakic and Lombroso). The formerly
mentioned domain is also termed the subpallial compartment, whereas the latter is referred to as
the pallial compartment. On the transcriptional level, the homeobox transcription factors distal-
less homeobox 1 (Dlx1) and distal-less homeobox 2 (Dlx2) were identified as GE markers in the
embryonic mouse forebrain, whereas the transcription factor NK2 homeobox 1 (Nkx2.1) has
been identified as a more specific marker of the MGE (Kimura et al.; Porteus et al.). These
transcription factors and the roles they play in specifying interneuron fate will be elaborated
upon in the discussion of interneuron specification.

32

Medial Ganglionic Eminence
As was previously mentioned, the MGE serves as a source of interneurons in the
developing cortex (Lavdas et al.). Neurons from the MGE posses their own “signature”, so to
speak: these neurons typically express GABA but are negative for expression of calretinin (CR),
which is a calcium-binding protein (Lavdas et al.). With regard to absence of calretinin
expression, these observations imply that most calretinin-positive interneurons originate from
spatially and/or temporally independent sources from that mentioned here. A small portion of
these GABA-positive, calretinin-negative neurons express the protein reelin. In addition to the
transcription factor Nkx2.1, the LIM-homeobox gene Lhx6 is a marker of MGE cells but is
specific for migrating MGE neurons (Lavdas et al.). Lhx6 is therefore hypothesized to play
some sort of role in the migration of cells from the MGE, although this has yet to be elucidated.
A large portion (approximately 60% of cortical interneurons in mice and rats) of MGE
interneurons also contains either PV or SST (Butt, Fuccillo, et al.; Valcanis and Tan; Wichterle,
Turnbull, et al.; Xu et al.). The fact that MGE cells express either PV or SST enables
categorization of MGE-derived neurons into two separate neurochemical groups that are also
unique in their physiological characteristics (Gonchar and Burkhalter; Kawaguchi and Kubota).
It has been agreed upon that the between the ganglionic eminences, the MGE is the primary
source of GABAergic interneurons during neurogenesis. Several studies have shown that MGE
cells possess a large migratory capacity, both in vitro and in vivo (S. A. Anderson, O. Marin, et
al.; Wichterle, Garcia-Verdugo, et al.; Wichterle, Turnbull, et al.).

33

Caudal Ganglionic Eminence
The caudal ganglionic eminence has been shown to be the second-greatest contributor of
interneuron progenitors that then migrate to the cerebral cortex (S. A. Anderson, O. Marin, et al.;
Nery, Corbin and Fishell; Nery, Fishell and Corbin). Miyoshi and colleagues utilized a genetic
fate mapping approach to corroborate the finding that the CGE is a source of cortical
interneurons (Miyoshi, Hjerling-Leffler, et al.). The CGE itself is unique in that it is somewhat
of a “hybrid” of the MGE and LGE: similar to the MGE, the ventral-most CGE expresses
transcription factor Nkx2.1, and the dorsal region of the CGE expresses the transcription factor
Gsh2, which is required for proper patterning of the LGE (Corbin et al.). CGE-derived cells
ultimately become deep-layer cortical interneurons (Nery, Fishell and Corbin). Neurons from
the CGE are unique from the MGE in the markers that they express: many contain either PV or
SST (Nery, Fishell and Corbin). Regarding calretinin expression, later studies showed that
calretinin-positive interneurons are generated largely in the Nkx2.1-negative region of the dorsal
CGE (Butt, Fuccillo, et al.; Lopez-Bendito et al.). This was proven utilizing in utero isochronic
homotopic transplants (Butt, Fuccillo, et al.), as well as construction of a transgenic mouse line
(Lopez-Bendito et al.).

Minor and miscellaneous sources of cortical interneurons
Embryonic Preoptic Area
A very recent study by Gelman et al has demonstrated that in addition to the ganglionic
eminences, the embryonic preoptic area (POA) should also be considered a source of cortical
GABAergic interneurons (Gelman et al.). The POA is a region of the hypothalamus, and results

34

from this study suggest that this area contributes approximately 10% of all GABAergic
interneurons in the murine cerebral cortex.

Lateral Ganglionic Eminence
While it is largely agreed upon that the MGE and CGE serve as the primary source of
cortical interneurons in the developing rodent nervous system, the possibility of the LGE as a
third source has also been heavily debated. Results from several studies have allowed the
conclusion that the LGE is at most a minor contributor of interneurons (S. A. Anderson, O.
Marin, et al.; Wichterle, Garcia-Verdugo, et al.; Wichterle, Turnbull, et al.). However,
observations from a few studies do suggest otherwise: the previously mentioned 1999 study by
Sussel and colleagues reported that Nkx2.1 mutants, in which normal MGE tissue fails to form,
show a 50% reduction in cortical interneuron numbers relative to wild-type (Sussel et al.). If the
MGE was the origin of the majority of cortical interneurons, only a 50% reduction implies that
there are clearly other areas of the brain responsible for generating interneurons. More
convincing evidence has shown that at E15.5, the LGE-like region in Nkx2.1 mutants
demonstrates strong cellular migration to the developing cortex (S. A. Anderson, O. Marin, et al.;
Nery, Corbin and Fishell). BrdU labeling of neural progenitors also supports the notion of a
cellular migratory route from the LGE to the cortex during embryogenesis, although only a
portion of the BrdU labeled cells were also GABA-positive (S. A. Anderson, O. Marin, et al.).
Additionally, Jimenez and colleagues discovered that when the MGE is removed in explants
taken from rat embryos, cellular migration from the LGE to the cortex continued to be observed,
suggesting that the migrating cells are not MGE cells merely passing through the LGE (Jimenez
et al.). While the LGE does seem to be a valid source of GABAergic interneurons, the lack of

35

conclusive data on this subject can only mean that the LGE may only be considered a minor, if
not a negligible, source at best.

Rostral Migratory Stream (RMS)
The rostral migratory stream (abbreviated RMS) is often regarded as a source of
interneurons, specifically in the context of the calretinin-positive interneuronal subtype. In the
developing brain of some (but not all) organisms, the RMS is a migratory route whereby neural
precursors generated in the subventricular zone migrate to reach the olfactory bulb. When
migratory neurons reach their destination in the olfactory bulb, they undergo differentiation to
become GABAergic interneurons. At postnatal day 0, RMS cells are observed to positively
express calretinin when cultured on cortical feeder cells (Xu et al.). This observation has
sparked the theory that before migrating to their final destination in the olfactory bulb, RMS cells
may first migrate to the cortex during neurogenesis. This theory is supported by
immunohistochemical data in which staining for Dlx1, a marker for migratory RMS precursors,
positively labels cells that are actively migrating from the RMS to the cortex (S. Anderson et al.).
Similar IHC staining for PSA-NCAM marker in rabbits supports the findings by Anderson et al
(Luzzati et al.).

Dorsal White Matter
In vivo studies, as well as time-lapse imaging studies performed on cortical slices, have
surprisingly revealed early postnatal dorsal white matter to be another potential source of
GABAergic interneuron precursors (Riccio et al.). These cells were observed in this location
during the first postnatal week and found to express the marker Pax6, indicating their precursor

36

fate. Time lapse imaging shows that these cells migrate from the dorsal white matter to the
cortex, suggesting a new postnatal source of cortical interneurons.

Septal Region
Previously, findings from several studies suggested that the septal region (the medial
olfactory area) was considered an additional source of GABAergic interneurons in rodents (S.
Anderson et al.; Taglialatela et al.). However, a 2010 study conducted by Rubin et al has
disproved these findings: the germinal zones of the basal ganglia, rather than the septum, are
responsible for generating interneurons of the developing cortex (Rubin et al.).

Figure 4: Sagittal (top) View of the Embryonic Telencephalon, Showing the Major Origins of
GABAergic Cortical Interneurons.

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37

Chapter Eleven: Specification of Interneurons
There has been a recent push in the study of GABAergic interneurons to understand and
create a transcriptional network that regulates GABAergic interneuron development, migration
to the cortex, and ultimately maturation to the appropriate adult phenotype. To understand the
complexity of the transcriptional network and other candidate genes involved in the production
and patterning of interneurons in the mammalian cortex, it is most logical to begin mapping this
network at the early phases of neurogenesis, when neural progenitors are actively proliferating in
the neuroepithelium. There are factors secreted within the neuroepithelium that influence and
control the proliferation of these progenitors (O'Leary and Sahara), one of which is Sonic
hedgehog (Shh) (Echelard et al.; Ericson et al.). Importantly, one of Shh’s main functions is to
activate homeobox transcription factor Nkx2.1 (Sussel et al.), and it does so by acting through
Gli transcription factors (Yu et al.). Xu et al showed that reduced or absent Shh signaling results
in significantly reduced Nkx2.1 expression, and therefore a reduction in PV- and SST-positive
interneurons (Xu, Wonders and Anderson). Interestingly, restoration of Shh signaling in Shh
nulls allowed the rescue of Nkx2.1 expression and SST-positive interneuron production,
suggesting interneuron specification is a somewhat plastic process (Gulacsi and Lillien; Xu,
Wonders and Anderson; Yung et al.).
Nkx2.1 is a functional MGE marker that is expressed in the proliferative zone of the
MGE (Kimura et al.). This transcription factor plays a large role in governing cortical
interneuron specification, as approximately 70% of GABAergic cortical interneurons derive
from the Nkx2.1-expressing domain (Miyoshi, Hjerling-Leffler, et al.). When absent in mouse
embryos, normal MGE tissue fails to form, and a 50% reduction in cortical interneurons is
observed relative to wild type (Sussel et al.). Xu et al demonstrated that E18.5 Nkx2.1 null

38

mouse embryos whose cortices were dissociated and analyzed in vitro lacked SST-, PV-, and
NPY-positive interneurons compared to wild-type cortices (Xu et al.). These findings therefore
suggest that Nkx2.1 functions to specify not only MGE-derived interneurons, but other subtypes
as well. Of note, however, was that CR-positive interneurons were unaffected by Nkx2.1
deletion (Xu et al.).
Seeing as proper functioning of Nkx2.1 plays such an important role in interneuron
specification, studies have also been performed to investigate its upstream- or downstream-acting
factors. Activation of Nkx2.1 subsequently results in the repression of the transcription factor
Paired box protein 6 (Pax6) (Inoue, Nakamura and Osumi) in the ventral-most area of the
neuroepithelium; this begins at E9.5 (Corbin et al.). This period of embryonic development is
also the time point at which the GEs appear, and also the time point at which cortical
GABAergic interneurons can be fate mapped utilizing recombination methods (Miyoshi, Butt, et
al.). With regard to utilizing transcription factor expression as a means of demarcating the
anatomical boundaries of the ganglionic eminences, none other than Nkx2.1 has been found to
do so. Additionally, the roles of many other proteins such as fibroblast growth factor 8 (FGF8),
homeobox protein SIX3, and bone morphogenetic protein (BMP), have all been investigated in
their ability to influence Nkx2.1 function (Kobayashi et al.; Lupo, Harris and Lewis; Storm et
al.). BMPs have been shown to play a crucial role in maintaining neural stem cell fate and
maturation, and, among many other functions, also play a part in inducing neurogenesis during
embryonic forebrain development (Bond, Bhalala and Kessler). BMP7 activity, for example, has
recently been shown to induce pSmad signaling and phosphatidylinositol 3-kinase- (PI3K)
dependent mechanisms that subsequently mediate the specification of spinal neurons (Perron and

39

Dodd). BMPs, because of the numerous roles they play in development, remain good candidates
for maintaining the specification of cortical interneurons.
Much is known about the local transcriptional network of the ventral ventricular zone;
several research groups have collectively determined that there are actual three transcriptionally
unique zones containing proliferating neural progenitors. In the literature these three zones are
referred to as the preoptic area (POA), medial ventricular zone, and the sulcal region. The three
key players (TFs) in patterning these three areas are: Nkx2.1 (TF expressed in the MGE), Dlx1
(GE marker), and homeobox protein Nkx6.2. All three factors are expressed in the POA, cells
within the medial VZ express only Nkx2.1, and both Nkx2.1 and Nkx6.2 are expressed in the
sulcal region (Stenman, Wang and Campbell). The family of Dlx homeobox genes is thought to
possess dual roles in the specification of interneurons as well as influencing their migratory
capabilities (S. A. Anderson, D. D. Eisenstat, et al.; S. A. Anderson, O. Marin, et al.; Cobos,
Borello and Rubenstein). Dlx1 and Lhx6 are expressed by migratory interneurons during the
early stages of neural development, but as development progresses only certain subgroups of
neurons express these transcription factors (Cobos et al.). PV-positive interneurons continuously
express Lhx6 throughout development, but later down-regulate Dlx1 expression, whereas CR-
positive interneurons continuously express Dlx1, even after birth (Cobos et al.). Neurons
belonging to the SST-positive interneuronal subtype express either only Dlx1 or only Lhx6, and
another percentage of SST-positive neurons expresses both transcription factors. Several groups
have carried out mutational analyses with regard to the Dlx genes. Cobos and colleagues
demonstrated that Dlx1 knockout mice possess significantly reduced numbers of SST- and CR-
positive neurons in the adult cortex and interestingly, present with epileptic behavior (Cobos et
al.). However, CR-positive interneurons are absent in Dlx1/2 null cortical cultures, implying that

40

this transcription factor also plays a role in specifying progenitors toward this particular subtype
(Xu et al.). Additionally, Dlx5/6 knockout mice possess reduced numbers of PV+ neurons (Y.
Wang, C. A. Dye, et al.). With regard to Nkx6.2, Sousa and colleagues have shown that its
expression in the sulcal region is variable and wanes as embryogenesis progresses (Sousa et al.),
and Flandin et al have most recently deduced that this transient pattern of expression is most
likely due to Lhx6/8-dependent of Shh induction of Shh in the mantle (Flandin et al.).
Additionally, high levels of Oligodendrocyte transcription factor 2 (Olig2) expression serve to
define the Nkx2.1 domain, with Olig2 protein expression occurring dorsally of the sulcal region
(Miyoshi, Butt, et al.).

Specification of Nkx2.1-Independent Interneuron Populations
While the Nkx2.1-positive population of neurons makes up the majority of all
GABAergic interneurons in the mature cerebral cortex, the remaining population of interneurons
is derived from a domain that is independent of the Nkx2.1-expressing domain. Relatively little
is known about these areas within the neuroepithelium that do not express Nkx2.1. Analyses of
previous studies (Anastasiades and Butt) have enabled a potential mechanism by which both
Nkx2.1-derived and Nkx2.1-independent GABAergic interneuron populations are generated
following the appearance of the GEs. When interneurons are first generated at approximately
E9.5, Nkx2.1 expression is the most influential. As development progresses Nkx6.2 expression
is then up-regulated (Flandin et al.; Xu, Roby and Callaway), in which a population of SST-
positive interneurons are generated. Nkx2.1 is also functioning at this time to repress the
homeobox gene Gsh2, whose expression would effectively cause inappropriate neuronal fate
switches (Butt, Sousa, et al.; Xu, Roby and Callaway). The CoupTF transcription factor family

41

now becomes a player in the game: expression occurs in the ventricular zone near the sulcal
region, and, along with a decrease in Nkx6.2 expression, a population of non-Nkx2.1
interneuronal subtypes is then generated (Miyoshi, Hjerling-Leffler, et al.; Sousa et al.). Because
cortical interneuronal subtypes that are derived from non-Nkx2.1-expressing domains are less
well studied, candidate transcription factors are currently being researched to understand how
these populations are specified. Among these transcription factors are Prospero homeobox
protein 1 (Prox1) (Srinivasan et al.), Mash1 (a proneural gene that induces the expression of
Dlx1/2) (Long et al.; Petryniak et al.; Schuurmans and Guillemot), several Sox genes (Ekonomou
et al.; Kan et al.), and Sp9, which is a regulator of Fibroblast growth factor (Fgf) signaling
(Kawakami et al.). Additionally, a group of transcription factors belonging to the CoupTF
family have recently been shown to play a role in the specification as well as migration of
cortical interneurons not governed by Nkx2.1 (Kanatani et al.; Lodato et al.). The transcription
factor CoupTF1 (NR2F1), for example, is expressed separately from the Nkx2.1 domain after
E13.5 and is thought to play a role in governing interneuron specification (Miyoshi, Hjerling-
Leffler, et al.). While this cohort of interneurons makes up a small percentage of the total
GABAergic interneuron population, the exact mechanism by which all interneuronal subtypes
are specified has yet to be elucidated.

Time Course of Interneuron Specification
The question of whether or not interneurons are specified (and therefore committed to
their ultimate fates) before or after they exit the cell cycle and migrate to their final destinations
has been highly debated. (The role that the neural precursor cell cycle plays in regulating
interneuron production is extensively reviewed by Ross (Ross). A few fate-mapping studies of

42

GABAergic interneuronal progenitors have investigated whether cells encounter specification
factors or signals during migration. In one study, MGE and CGE progenitors were plated on
feeder layers and their respective fates were determined 2-4 weeks after plating (Xu et al.). In a
subsequent study, the physiological characteristics of genetically labeled MGE and CGE cells
were assessed after in utero transplantation back into the MGE and CGE, respectively (Butt,
Fuccillo, et al.). Results from both studies allowed the conclusion that both MGE- and CGE-
derived interneurons are specified before cell cycle exit and migration to their final destinations;
evidence largely discounts the possibility that signals encountered during migration influence
specification, although it has not been completely ruled out (Butt, Fuccillo, et al.; Valcanis and
Tan; Xu et al.).
Following their exit from the cell cycle, maturing interneuron precursors destined for the
neocortex and those destined for the striatum begin to differ in their transcriptional expression
profiles. Future cortical interneurons, which will ultimately form a network with glutamatergic
pyramidal projection neurons of the cerebral cortex, begin to down-regulate Nkx2.1 upon exiting
the cell cycle (Nobrega-Pereira et al.). This down-regulation effectively results in the up-
regulation of factors that will influence the migratory route of this group of interneurons toward
the cortex (Marin et al.). Cortical interneuron precursors then begin to express Lhx6 in the
subventricular zone (Alifragis, Liapi and Parnavelas; Du et al.; Grigoriou et al.; Sussel et al.).
Only PV- and SST-positive cells express Lhx6 as it is downstream of Nkx2.1 (Du et al.; Fogarty
et al.; Liodis et al.; Zhao et al.), and expression is maintained throughout migration to the cortex
(Lavdas et al.). Deletion of Lhx6 confirms that it is downstream of Nkx2.1, as a similar
phenotype (decreased PV- and SST-positive cells) to Nkx2.1 knockouts is observed.

43

Figure 5: General Schematic of GABAergic Cortical Interneuron Specification.

Candidate Factors for Interneuron Specification
Most recently, various studies have investigated the roles that candidate molecules and
factors may have in specifying the GABAergic interneuron population. One study found that a
population of cortical interneurons derived from both the dorsal LGE and the dorsal CGE
express the transcription factor Sp8 (Ma et al.). These interneurons represent approximately
20% of all adult cortical GABAergic interneurons and are relatively later-born neurons, so they
migrate to the cortical layers during the early postnatal stages (Ma et al.). An additional study
utilized BrdU incorporation to show that Sp8 is actually required for the production of PV-
positive interneurons located in the olfactory bulb (Li et al.). Paired-like domain transcription
factor Pitx2 is a paired-like homeodomain transcription factor that is expressed in post-mitotic
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44

neurons. A 2011 study has shown that a population of Pitx2+ neurons in the stratum griseum
intermedium (SGI) (in the dorsal midbrain) was actually found to be GABAergic neurons.
While this population of neurons does not pertain to cortical GABAergic interneurons per se,
these findings suggest that Pitx2 is another transcription factor that may be added to the list of
candidates influencing GABAergic neuron specification and differentiation (Waite et al.).
Chatzi et al have recently discovered that retinoic acid, a vitamin A metabolite, may play a role
in regulating GABAergic interneuron production (Chatzi, Brade and Duester). Retinaldehyde
dehydrogenase mutant mouse embryos with the inability to generate retinoic acid demonstrated a
significant deficiency in GABAergic interneurons (Chatzi, Brade and Duester). Interestingly,
factors that play a role in preventing interneuron specification have also been identified. A LIM
complex known as Isl1-Lhx3 drives motor neuron fate in the embryonic spinal cord; RNA-seq
data show that these proteins induce the expression of genes directly involved with the
production of motor neurons (S. Lee et al.). In doing so, however, key interneuron genes are
suppressed, thereby revealing a mode of negatively regulating interneuron specification and
differentiation.

Importance of Signaling Pathways in Interneuron Specification
Recent research on Ryk (abbreviated for Related to receptor tyrosine kinase), which
serves as a receptor of the Wnt signaling pathway and is required for the stimulation of neurite
outgrowth (Lu et al.; Lyu, Yamamoto and Lu), has demonstrated that it is involved in promoting
GABAergic interneuron production, while simultaneously repressing oligodendrocyte fate in the
ventral telencephalon (Zhong et al.). More specifically, Ryk acts to negatively regulate Olig2, a
factor involved in oligodendrogenesis, and induces expression of interneuron fate marker Dlx2.

45

Findings from this study open up the possibility that the Wnt signaling pathway plays a role in
the specification and/or maintenance of interneuron fate. One study has shown that the non-
canonical Wnt signaling pathway, specifically Wnt5a, plays a role in the development of
olfactory bulb interneurons both in vitro and in vivo (Pino, Choe and Pleasure).
The involvement of signaling pathways in the specification of cortical interneuron fate is
a topic that has been extensively researched, one being the Shh signaling pathway. Shh acts via
the Gli transcription factors, one of which is Gli3 (Yu et al.). Gli3 is required for the
maintenance and specification of cortical progenitors. Conditional deletion in mice, combined
with the techniques of birthdating and in utero electroporation, have revealed that Gli3 is
necessary for specifying cortical interneuron fate and interestingly, for ensuring that progenitors
are actively engaged in the cell cycle (H. Wang, G. Ge, et al.). In addition to playing a role in
the development of cortical interneurons, the Shh signaling pathway is also required for the
regulation of thalamic interneuron identity. Jeong and colleagues have proposed a model
whereby Shh signaling from spatial and temporal domains in the diencephalon is involved in the
development of certain classes of thalamic interneurons (Jeong et al.).
Zou et al show that the Erk5 signaling pathway may play a role in regulating
neurogenesis in the olfactory bulb: deletion of Erk5 in mouse neural stem cells results in a
significant decrease in GABAergic interneuron number in the olfactory bulb (Zou et al.). While
these results do not pertain to cortical GABAergic interneurons, one must not discount the
possibility that Erk5 is yet another signaling pathway that has the potential to influence cortical
interneuron development.
As was previously discussed, BMPs have been shown to play a crucial role in
maintaining neural stem cell fate and maturation, which opens up the possibility that the BMP

46

signaling pathway is somehow involved in the specification of cortical interneurons (Perron and
Dodd). Additionally, a study performed in zebrafish larvae showed that altering dopamine D
2

receptor activity modulates Akt protein kinase signaling, and inhibition of Akt signaling leads to
a reduction in the GABAergic interneuron population in the brain (Souza, Romano-Silva and
Tropepe), thereby suggesting the necessity of Akt signaling in promoting interneuronal
specification.

Differences in Cortical Interneuron Origin and Specification Between Rodents and
Primates
Studies in both primates and humans have determined that differences do exist between
rodents and primates with regard to the origins and migratory routes of GABAergic interneurons.
This will be briefly discussed, but for more detail refer to the review by Petanjek et al (Petanjek,
Kostovic and Esclapez).
A few studies have shed light on the fact that the majority of primate GABAergic
interneurons, in contrast to rodent cortical interneurons, may not actually originate solely in the
GEs (Letinic, Zoncu and Rakic; Yu and Zecevic). In fact, cortical interneurons in primates have
also been shown to have their origins in the proliferative zones of the dorsal telencephalon
(Fertuzinhos et al.; Letinic, Zoncu and Rakic; Petanjek, Berger and Esclapez; Rakic and
Zecevic). Letinic and colleagues utilized retroviral labeling in organotypic slice cultures to
demonstrate that there are two independent lineages of cortical interneurons in the fetal human
forebrain (Letinic, Zoncu and Rakic). However, whereas it was previously mentioned that the
GE is the primary source of rodent interneurons, this study demonstrated that in one lineage of
cortical interneurons in the fetal human forebrain, only 35% of the interneuron population in the

47

proliferative ventricular zone and subventricular zone originates from the GE (Letinic, Zoncu
and Rakic). To support these findings, the same results were reported in the macaque monkey
(Macaca rhesus, Macaca fascicularis) (Petanjek, Berger and Esclapez).
It is suspected that GABAergic cortical interneurons in primates are largely generated
locally, rather than in the GE (as in the case of their rodent counterparts). This is based on the
lack of Mash1 expression in the GE, as Mash1 is a transcription factor involved in the
specification of GABAergic neurons (Petanjek, Kostovic and Esclapez). At E47-55, no Mash1-
positive cells were found in a region very close to the GE, whereas positive expression was
observed in the VZ/SVZ at that developmental time point.
In addition to differing locations of interneuron origin between species, it is also
important to realize that the transcription factors expressed by rodent interneurons and
interneuronal precursors may not necessarily overlap with the transcriptional network that
governs the development of human GABAergic interneurons. Research to better characterize
human interneurons is underway. One such study investigated the expression of transcription
factors Nkx2.1, Dlx1/2, Lhx6 and Mash1 in human fetal forebrains during the first half of
gestation (Zecevic, Hu and Jakovcevski). All transcription factors were expressed in both the
GEs and the ventricular/subventricular zones, and expression was maintained up to 20
gestational weeks. Collectively the data suggest that cortical interneuron populations exist in
multiple locations, both ventrally (as described in rodents) and dorsally in the VZ/SVZ (Zecevic,
Hu and Jakovcevski). Findings from a previous study utilizing cryosections and in vitro data
also support the existence of multiple sources of cortical interneuron progenitors in the
developing human brain (Jakovcevski, Mayer and Zecevic). The greater complexity

48

characterizing progenitor populations most likely reflects the higher brain functioning
characteristic of humans compared to other organisms.

Clinical Implications of GABAergic Interneuron Specification
Research on GABAergic interneurons is often carried out with the hopes of
understanding mechanisms behind neurological disorders, one being Tuberous sclerosis complex
(TSC). TSC is a genetic disease characterized by neurologic and psychiatric symptoms
including epilepsy, developmental delay and autism. The mechanisms behind TSC have not yet
been elucidated, but studies performed on a gene known as Tsc1 have shown that it is essential
for GABAergic interneuron development, function, and potentially interneuronal migration as
well (Fu et al.). Tsc1 conditional knockout mice were generated and in addition to having
impaired growth and survival, were found to possess a reduced number of GABAergic
interneurons in the cortex, as well as a reduction of specific GABAergic subtypes found (Fu et
al.). Along the lines of neurological disorders, Disrupted-in-Schizophrenia-1 (DISC-1) is a gene
whose mutation is implicated in numerous psychiatric disorders, and has been shown to possess
roles in neuronal proliferation and differentiation in the cerebral cortex (Brandon et al.). DISC-1
is necessary for the migration of MGE-derived cortical interneurons (Steinecke et al.). DISC-1
knockdown via in utero and ex utero electroporation significantly hindered the migration of
these interneurons.

49

Chapter Twelve: Migration to the Cerebral Cortex
One of the most interesting and essential characteristics of GABAergic interneurons,
regardless of their subtype, is that once they are generated and specified in the ganglionic
eminences, these interneurons face the task of migrating to their ultimate destinations in the adult
brain (Tanaka, Oiwa, et al.). Among these final destinations are the cerebral cortex, amygdala,
striatum, hippocampus, and olfactory bulb. It has been observed that GABAergic interneurons
first begin their migration at E12.5 in rodents, a time point that also happens to correspond with
the early stages of neurogenesis (Corbin, Nery and Fishell; Marin and Rubenstein "A Long,
Remarkable Journey: Tangential Migration in the Telencephalon"; Wonders and Anderson).
Migration is, for the most part, complete by birth, with exception of the RMS; the migration
route that neurons in the RMS take to reach the olfactory bulb will not be covered. The most
well-studied and well-characterized route of interneuron migration, which is the route neurons
take to reach the cerebral cortex, will be discussed here.
After generation and specification in the ganglionic eminences, interneurons must make
their way to the cerebral cortex, and take two possible approaches to do so (Marin et al.). One of
these two routes is designated as an outer route, which occurs along the marginal zone; the
second has been coined an inner route, which occurs along the subventricular zone.
Visualization of migratory neurons via live imaging studies have shown that once they reach the
cerebral cortex, these neurons undergo a secondary type of movement before reaching their final
destination in the cortical plate (Ang et al.; Tanaka, Maekawa, et al.; Tanaka, Yanagida, et al.).
One study investigated the migratory route of interneurons destined for either the superficial
preplate layer of the cortex or a deeper cortical layer in the intermediate zone (Antypa et al.).
Microarray analysis data showed that several novel genes of interest were up-regulated in one

50

migratory stream but not the other, suggesting that the routes interneuron precursors take are
dependent on the ultimate destination in the cortex. It has also been shown that in the case of
reelin- and calretinin-positive interneurons, neuronal activity is required before postnatal day 3
for correct migration to the cortex (De Marco Garcia, Karayannis and Fishell). However, rather
than elaborating upon the migration pattern itself that interneurons take, the factors that influence
the interneuronal migratory route will be addressed.

Motogens
Motogens are secreted factors that influence newly specified interneurons in their ability
to migrate (Marin and Rubenstein "Cell Migration in the Forebrain"). Hepatocyte Growth Factor
(HGF) is a mitogen that was discovered to regulate the migratory abilities of subpallial-derived
cortical interneurons. HGF has been found to be required for interneuron migration, as mu-PAR
nulls (mu-PAR is a required component of the HGF pathway) demonstrate significant deficits in
interneuron migration to the cortex (Powell et al.; Powell, Mars and Levitt). In their 2003 study
Powell et al also presented the finding that HGF loss of function has an effect on some, but not
all, subsets of interneurons (Powell et al.). In addition to HGF, the neurotrophic factors
Neurotrophin-4 (NT4) and Brain-Derived Neurotrophin Factor (BDNF) have also been found to
serve as motogens for migratory interneurons (Brunstrom et al.). Similar to HGF, loss of
functioning neurotrophin receptors results in a decrease in interneuron numbers in the cortex
(Polleux et al.).

51

Chemorepellents and Chemoattractants
Whereas motogens affect the ability of interneurons to migrate, chemorepellants and
chemoattractants serve to provide migratory cells with the information about which direction to
migrate. Wichterle and colleagues have shown through cell culture studies that in the
telencephalon, the GEs exert a repulsive force on interneurons, whereas the cerebral cortex
exerts an attractive force on them (Wichterle, Alvarez-Dolado, et al.). This crucial study
therefore allowed the conclusion that chemorepellants are primarily expressed in the subpallium.
To date, the Semaphorin (Sem) family, especially Sem3A and Sem3F, are a prime example of
chemorepellants (Marin et al.; Tamamaki et al.). Sem3A and 3F are both expressed in the LGE,
consistent with the notion that chemorepellants are located in the subpallium. Techniques such
as in vitro migration assays and in vivo loss of function approaches have allowed the realization
that molecules of the Sem family, along with their receptors, are required for migration of
interneurons to the cortex. In particular, the family of Roundabout receptors, more commonly
referred to as Robo receptors, are regarded as regulators of semaphorin signaling, and one
research group has clarified the mechanism by which Robo1 regulates semaphorin signaling.
Robo1, expressed by cortical interneurons, directly interacts with neuropilin receptor 1 (Nrp1)
expressed in striatal mantle cells. The binding of Robo1 to Nrp1 serves to modulate semaphorin
signaling in the developing forebrain, which subsequently directs the migration of interneurons
into the cortex. This study demonstrated that loss of Robo1 function in mice retards semaphorin
signaling and therefore hinders interneuron migration (Hernandez-Miranda et al.).
Whereas chemorepellants are located in the subpallium, chemoattractants exert an
attractive force on interneurons and are present in the pallium. Glial cell-derived neurotrophic
factor (GDNF) and the family of Neuregulins (NRG) are the most well-studied and best

52

examples of chemoattractants to GABAergic interneurons (Flames et al.; Pozas and Ibanez). In
addition to serving as chemoattractants, GDNF and NRG also function to regulate cell
proliferation, survival, and differentiation throughout the nervous system. Via studies in vitro,
Neuregulin1 (NRG1) in particular is known to act as a chemoattractant for MGE-derived cells
(Flames et al.). Research has also been performed on the NRG receptor ErbB4, which is
expressed on migratory interneurons (Yau et al.). In mature, conditional ErbB4 mutants, a
reduced number of cortical interneurons is observed, indicating the requirement of NRGs in the
interneuronal migratory route to the cortex (Flames et al.). More recently, chemokines have also
been reported to function as chemoattractants for MGE-derived interneurons, particularly
chemokine receptors type 4 and 7 (CXCR4 and CXCR7) (Sanchez-Alcaniz et al.; Y. Wang, G.
Li, et al.).
It is clear that when the tangential migration route of interneurons destined for the cortex
is altered or genetically disrupted, neurological deficits and defects are likely to occur. One
research group utilized a Foxc1 hypomorph mouse model (named Foxc1hith/hith) with
meningeal defects and observed significantly impaired interneuron migration (Zarbalis et al.).
Findings from this study suggest that meningeal defects may be the primary cause for the
reduced capacity of migration, and interestingly, that the chemokine ligand 12 (Cxcl12) may
potentially be the major player ensuring interneuronal migration (Zarbalis, Choe et al. 2012).
Understanding whether or not interneurons derived from specific domains are
predetermined to migrate along particular routes to their final destinations has yet to be officially
determined. Interneurons derived from the POA express EphrinB3, which acts as a repellent that
prevents these interneurons from migrating into the deeper cortical layers and enables them to
take the superficial route (Zimmer et al.). On the other hand, interneurons derived from the

53

MGE are influenced by Ephrin4A, which enables their migration into deep layers rather than
superficial layers of the cortex (Zimmer et al.). EphrinB3 and 4A therefore act to ensure that
POA- and MGE-derived interneurons will migrate independently of one another.

Neurotransmitters
Although relatively not as well studied, neurotransmitters are also thought to play an
important role in interneuron migration. GABA, glutamate, and dopamine are all present in the
embryonic brain, although the role they play has not yet been elucidated (Crandall et al.; Manent
et al.; Represa and Ben-Ari).

Lhx6
Of particular interest to the migration route of cortical interneurons is the LIM
homeodomain transcription factor Lhx6. MGE-derived interneurons that are actively migrating
to the cerebral cortex express Lhx6 (Gong et al.; Lavdas et al.). Moreover, Lhx6 is also
expressed in most PV- and SST-positive cortical interneurons in mice (Cobos et al.). Utilization
of Lhx6 knockdown using RNAi hindered the migration of cortical interneurons, lending strong
support to the postulation that the Lhx6 gene somehow plays a role in migratory capability of
interneurons (Alifragis, Liapi and Parnavelas).

54

Conclusion
Although asymmetric cell division plays a role in the developmental processes of many
organisms, it has been best studied in the context of neurogenesis in Drosophila neuroblasts.
While the molecules and proteins that play roles in setting up cortical polarity and spindle
orientation have been heavily studied and documented, the utilization of neuroblasts as a model
to study uncontrolled stem cell self-renewal, and ultimately, tumorigenesis, is relatively new. It
is clear that impinging upon any of these components of the asymmetric division machinery will
have dire consequences. Transplantation models such as that utilized by Laurenson et al are
likely to provide insights into the connection between asymmetric division and tumorigenesis.
A “big picture” approach to understanding the carefully controlled balance between
neural stem cell self-renewal and differentiation has utilized transgenic RNAi on the genome-
wide level to identify over 600 genes that may control this self-renewal vs. differentiation switch
(Neumuller et al.). Knockdown of key genes, such as transcription factors Lola, Ssrp and Barc,
results in defective neuroblast lineages. The identification of genes in this study should be
further studied with the objective of creating functional gene networks that interact or influence
asymmetric cell division machinery.
Despite the advances that have been made in this field of research, there are many
questions that have yet to be answered. For one, the exact time point at which neuroblasts re-
enter the cell cycle at the end of the embryonic stage remains unclear. Additionally, one must
consider the fact that asymmetric cell division in mammalian systems opens a whole new door of
complexity: although there may be a high degree of conservation between Drosophila and
mammalian homologs, the roles that these mammalian homologs play may be not at all similar to
that of the cell fate determinants and proteins in Drosophila. Gaining an understanding of the

55

similarities and differences between Drosophila neuroblasts and mammalian neural progenitors
may hold the key to understanding and/or treating various neurological degenerative disorders.
Just as many advances are being made in the field of asymmetric cell division, the same
can definitely be said about GABAergic interneurons of the mammalian cerebral cortex. The
population of GABAergic interneurons in the cerebral cortex is a clearly a diverse one,
comprised of many different subtypes and functions. While each interneuronal subtype is
characteristically unique with regard to function, electrophysiology, immunohistochemical
profile, axonal targeting and firing pattern, the overlapping features between particular subtypes
makes the method of categorizing each subset of GABAergic interneurons quite challenging.
Most recent undertakings in the study of cortical interneurons have been concerned with
constructing the transcriptional network of genes that are involved in the specification of these
interneurons, in pre-migratory stages within the subpallium as well as during the migratory phase
of interneuron precursors. Additional investigations must be performed in order to understand
the specific transcription factors and signaling pathways involved in the specification of
interneuronal fate. More specifically, studies must be undertaken to understand how the Nkx2.1-
independent interneuron lineage is specified before migration of these interneuronal progenitors
to the cerebral cortex. Additionally, little is known about the differential specification of PV-
and SST-positive subgroups within the Nkx2.1-expressing lineage of interneuronal progenitors.
The emergence of cutting edge genetic technologies should be utilized to target specific
interneuronal subtypes and understand what key players will determine their fate and function.
On a broader level, investigations must also be carried out to understand the differences in origin
and specification of rodent vs. human GABAergic interneurons. Understanding the mechanisms
behind human GABAergic interneuron specification may enable a large step forward in treating

56

various neurological disorders linked to alterations/dysfunctions in interneuron populations. An
increased understanding of the mechanism(s) by which GABAergic interneurons are able to
integrate properly and seamlessly into the cortex after migration is complete is also warranted.

57

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Abstract (if available)

Abstract On the most basic level, an asymmetric division is a developmental process that produces two daughter cells, each possessing a different identity or fate. Progenitor cells known as neuroblasts undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell. There are several features of asymmetric division in Drosophila melanogaster that make it a very complex process. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role of mitotic spindle orientation, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. A number of neuronal types and subtypes develop upon the completion of neurogenesis. One type of neuron that will be discussed at length is the GABAergic interneuron of the cerebral cortex. GABAergic interneurons are inhibitory neurons of the nervous system that are so named due to their release of the neurotransmitter gamma-aminobutyric acid (GABA). The developmental origins of GABAergic interneurons will be discussed, as well as factors that influence the migration routes that these interneurons must take in order to ultimately localize in the cerebral cortex. A number of recent findings concerning the transcriptional network of genes and candidate genes that play a role in the specification and maintenance of GABAergic interneuron fate will be discussed. Gaining an understanding of the different aspects of cortical interneuron development and specification, especially in humans, has useful clinical applications that may serve to treat various neurological disorders linked to alterations in interneuron populations.

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Asset Metadata

Creator Kelsom, Corey Sayuri Kauai (author)

Core Title Asymmetric cell division during neurogenesis, and the mechanisms behind GABAergic cortical interneuron development and specification

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 11/19/2012

Defense Date 09/20/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag asymmetric,cortical,GABA,interneuron,neuroblast,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Hacia, Joseph G. (committee member), Lu, Wange (committee member)

Creator Email ckelsom@gmail.com,kelsom@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-116106

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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