Investigation of Common Bases of Sympathetic Nervous System and Neuroblastoma Development

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FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences (Cancer Biology)

Investigation of common bases of sympathetic nervous system and neuroblastoma development

Submitted by: Huilin Shi

In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences

Examination Committee Signature/Date

Major Advisor: Han-Fei Ding, Ph.D.

Academic William Maltese, Ph.D. Advisory Committee: Manohar Ratnam, Ph.D.

Ivana de la Serna, Ph.D.

Zi-jian Xie, Ph.D.

Hongjuan Cui, Ph.D.

Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D.

Date of Defense: April 30, 2009

The common basis of sympathetic nervous system

and neuroblastoma development

Huilin Shi

Cancer Biology Track

College of Medicine

University of Toledo

2009

ACKNOWLEDGMENTS

I would like to sincerely thank my major advisor, Dr. Han-Fei Ding, for his patience, encouragement, support and incredibale guidance in my five years Ph.D. study.

His perpetual enthusiasm, abundant knowledge, outstanding scientific thinking and hard-

working attitude have always motivated me to make progress in the research and in the

future career.

I would like to especially thank my co-advisor Dr. William Maltese for his

supervision, guidance and invaluable support in the fifth year of my study and helping me

complete the work in manuscript two.

I would like to express my gratitude to my committee members: Dr. Manohar

Ratnam, Dr. Zi-Jian Xie, Dr. Ivana de la Serna and Dr. Hongjuan Cui, for their precious and extensive personal and professional guidance in my study. They teach me diverse thinking styles in science and in real life that make me more open-minded.

I would like to acknowledge Dr. William Gunning for teaching me solid knowledge of microscopy and making me adequate for doing the research in my first

manuscript while providing pathological supplies in my research. I would also like to

thank Dr. David Giovannucci and Dr. Andrea Nestor for their help in confocal

microscopy. I am grateful to Dr. Ming Zhang for his advice and help in the histological

analysis of my first paper.

I would also like to give my appreciation to all the members in Dr. Maltese’s lab:

Dr. Jean Overmeyer, Ashley Young, Dr. Aparna Kaul, Kristen Koterba and Haymanti

Bhanot, for their kindness, patience and generous technical assistance.

I would like to thank Jane Ding for her hard work and providing us an excellent

research support. I’m very grateful to other lab colleagues, Goleeta Alam, Dr. Baochun

Zhang, Dr. Zhe Wang, Dr. Jun Ma, Liqun Yang, who have supported me with technical

assistance, encouragement and friendship.

I would like to give my special thankfulness to my English teacher and friend, Dr.

Andy Chermak and his wife Dr. Shelley Green for their warm support in my personal life

and helping me dissolving a lot of language problems.

At last, I would like to give my gratefulness to my parents for their complete care

and understanding.

iii

TABLE OF CONTENTS

Acknowledgements ………………………………………………………...... ii

Table of Contents …………………………………………………………………... iv

Introduction ………………………………………………………………………… 1

Literature ……………………………………………………………………………. 8

Manuscript One ……………………………………………………………………. 52

Nestin expression defines both glial and neuronal progenitors in postnatal

sympathetic ganglia

Manuscript Two ……………………………………………………………………. 90

GATA3 regulation of human neuroblastoma stem cell activities

Summary/Discussion …………………………………………………………….... 148

Conclusions ……………………………………………………………………….. 152

Bibliography ………………………………………………………………………. 154

Abstract ……………………………………………………………………………. 195

INTRODUCTION

The sympathetic nervous system originates from ventrally migrating neural

crest cells that are derived from the dorsal neural tube, including sympathetic ganglia

and adrenal medulla. Two chains of sympathetic ganglion primordia are formed along

the both sides of the dorsal aorta in the early embryonic development. Through a series of differentiation processes, noradrenergic neuroblasts and glial progenitors are developed from neural crest cells and coalesce to form definitive sympathetic ganglia.

Sympathetic ganglia have been widely used as a model in studying the neurogenesis

(Schafer et al., 1997; Stanke et al., 1999; Tsarovina et al., 2004), such as mechanisms under the autonomic neuronal differentiation (Goridis and Rohrer, 2002; Howard,

2005), signals controlling the sympathetic ganglion development (Glebova and Ginty,

2005) and functions of ganglion cells (Hirst and McLachlan, 1984). In 1991, Hall and

Landis found that in the embryonic rat superior cervical ganglia (SCG), the

development of neuronal and glial cell precursors occurs with temporal differences.

Neurogenesis peaks around embryonic day 14.5 (E14.5) and is completed around

E17.5 (Hall and Landis, 1991). However, glial progenitor cells proliferate intensely

during E16.5 to E18.5 and thereafter continue proliferating but at a slower rate (Hall

and Landis, 1992). These previous studies propose the following questions: Are there

any neural progenitor cells in the postnatal sympathetic ganglia? What is the pattern

of gliogenesis and neurogenesis in the postnatal sympathetic development? Are there

any specific molecular markers to identify progenitor cells in the sympathetic ganglia?

To answer these questions, we investigated the proliferation and differentiation

status in the postnatal mouse SCG at different time points from newborn to two

months. Nuclear antigen Ki67 was used as a proliferation marker to detect

proliferating cells in SCG, which is expressed in the G1, S, G2 and M phases of the

cell cycle, but not in G0 (Sawhney and Hall, 1992). The differentiation process was

determined by bromodeoxyuridine (BrdU) tracing. The major action of BrdU is to

incorporate into DNA at S phase through acting as a thymine analogue and can

transfer to progeny cells during cell division (Matsuoka et al., 1990). Colocalization

of BrdU with different cell type markers in a sequential time course identifies cell

differentiation.

We found that Ki67-positive proliferating cells show a gradually decreasing

process during postnatal three weeks. The majority of Ki67-positive cells are

coexpressed with brain lipid-binding protein (BLBP), a glial cell marker labeling both

glial progenitors and fully differentiated mature glial cells. The expression level of

BLBP remains in a stable status during the postnatal period. Therefore, postnatal development in the mouse SCG is characterized by gliogenesis. We also use another glial cell marker, S100, to trace postnatal gliogenesis. S100 expression exhibits a gradually increasing pattern postnatally. Its expression starts to be detected around postnatal day 4 (P4), peaks around P18 and persists thereafter. During P7 to P18, a small number of Ki67 and S100 double-positive cells are observed which confirms that S100 specifically labels the mature glial cell group. We used tyrosine hydroxylase (TH) as a neuronal marker that is a specific marker for sympathetic neurons because it is the first and rate-limiting enzyme in synthesizing the catecholamine neurotransmitters. To our surprise, there are still a small percentage of

Ki67 and TH double-positive cells during postnatal week one. These observations

delineate different proliferation patterns of gliogenesis and neurogenesis in the

postnatal sympathetic ganglia. During the differentiation study by BrdU tracing, we

observed that a population of BrdU and BLBP double-positive cells can differentiate

into a group of BrdU and S100 double-positive mature glial cells, confirming the

dominant role of gliogenesis in postnatal sympathetic development.

We also identified a new glial progenitor cell marker, nestin, previously a neural progenitor cell marker in the central nervous system (CNS) and in neural crest cells (Lendahl et al., 1990; Lothian and Lendahl, 1997). The expression of nestin is highest at birth and gradually decreases during postnatal three weeks similar to the pattern of Ki67 expression. The majority of nestin positive cells are also Ki67 positive, which indicates that nestin-positive cells represent a progenitor proliferating cell population in postnatal sympathetic development. In the differentiation study, similar to Ki67, approximately 95% of the BrdU positive cells are co-stained with nestin.

BrdU and nestin double-positive cells are extensively coexpressed with BLBP and sparsely colocalized with TH which demonstrates that nestin is a marker for both glial and neuronal progenitors.

Our findings provide nestin as a molecular marker that defines the distinct

sympathetic progenitor cell population. The detailed characterization of the celluar

basis for the postnatal sympathetic development using different population markers

provides a foundation for the further investigation of genes and signaling pathways

that regulate the developmental process.

Neuroblastoma is the most common solid malignant tumor in infancy and

childhood which originates from the sympathetic nervous system, occurring in 3

sympathetic ganglia and adrenal medulla (Brodeur, 2003). 90% of children with this disease are diagnosed before 6 years of age (Schwab et al., 2003). Therefore the

tumorigenesis of neuroblastoma may represent an abnormal sympathetic development

in the embryonic or postnatal phase.

The cancer stem cell model reflects a new viewpoint on tumorigenesis. Cancer stem cells are cancer cells that generate tumors through their stem cell abilities – self

renewal and differentiation – and give rise to all the cell types found in a particular

tumor. These cells are proposed to persist in tumors as a distinct population to give

rise to new tumors and cause relapse and metastases. Since neuroblastoma is one of

the few cancers occurring in infancy and childhood, it is possible that neuroblastoma

originates from transformed neural crest stem cells or malignant sympathetic

progenitor cells that obtain the stem cell properties through mutations.

Neuroblastoma stem cells have been demonstrated to exist in neuroblastoma

tumors (Hirschmann-Jax et al., 2004; Walton et al., 2004). I-type (intermediate)

neuroblastoma cell lines have been proven to be one kind of neuroblastoma stem cells based on their self-renewal and differentiation ability. I-type neuroblastoma cells exhibit dramatically higher tumorigenicity in both soft agar assays and immunodeficient mice compared with N-type (neuroblastic) and S-type (non-neuronal, substrate-adherent) neuroblastoma cell lines (Cui et al., 2006; Spengler et al., 1997).

Therefore, it is important to investigate the mechanisms under the regulation

of neuroblastoma stem cells in order to better understand the origin and development

of this disease and establish effective clinical therapies targeting the root of

neuroblastomas. Of the thousands of regulators, we chose GATA3 as the one to

investigate its regulation in neuroblastoma stem cell activities. I chose GATA3 based

on following reasons:

First, GATA3 belongs to the GATA family that contains 6 members of zinc

finger transcription factors (GATA1 to 6) in vertebrates (Patient and McGhee, 2002).

GATA family members play important roles in vertebrate development. The mutation

and expression modulation of GATA family members can induce human diseases. A

GATA1 mutation results in dyserythropoietic anemia and thrombocytopenia (Nichols

et al., 2000). The HDR syndrome – hypoparathyroidism, deafness, and renal dysplasia

– is caused by a GATA3 mutation (Van Esch et al., 2000). A GATA4 mutation induces congenital heart defects (Garg et al., 2003). GATA3 plays an essential role in

the development of many tissues in vertebrates, including the Th2 cell differentiation, the development of the kidney, the inner ear, embryonic and pubertal mammary glands, adipocytes and neurons, et al. (Asselin-Labat et al., 2007; Grote et al., 2008;

Kouros-Mehr et al., 2008; Lillevali et al., 2006; Smith et al., 2002; Tong et al., 2005; van Doorninck et al., 1999; Yamashita et al., 2005).

Second, GATA3 is essential for normal sympathetic development. The bone

morphogenetic proteins (BMPs) secreted from cells of the dorsal aorta triggers the expression of downstream transcription factors – Phox2b, Mash1, Phox2a, dHand and

GATA3 in the neuronal progenitor cells and leads the cells to gain the characteristics of differentiated sympathetic neurons (Howard, 2005). GATA3 controls sympathoadrenal differentiation by mutual modulations with other sympathetic regulators (Moriguchi et al., 2006). In the mouse sympathetic system, GATA3 knockout embryos die by E11 due to noradrenaline deficiency and loss of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) (Lim et al., 2000). Loss of

GATA3 results in the shrinkage of the ganglion size and induces apoptosis in the

sympathetic ganglia and adrenal glands (Moriguchi et al., 2006; Tsarovina et al.,

2004).

Third, GATA3 also plays different roles in tumorigenesis in multiple tumors,

such as T cell acute lymphoblastic leukemias (T-ALL), breast and pancreatic cancers,

cervical and esophageal carcinomas (Gulbinas et al., 2006; Minegishi et al., 1997;

Shiga et al., 1993; Steenbergen et al., 2002; Usary et al., 2004).

Since GATA3 is an essential regulator in the vertebrate development and plays

an important role in the sympathetic development and tumorigenesis, we proposed the

hypothesis that GATA3 has important functions in regulating the neuroblastoma stem

cell activities. We use BE(2)-C cells as an I-type neuroblastoma stem cell model to

investigate the role of GATA3 in this system.

We found that loss of GATA3 induces the increased tumorigenicity of BE(2)-C

cells, whereas overexpression of GATA3 decreases the tumorigenic ability of BE(2)-

C cells as evidenced by soft agar assays. The clonogenic assay, MTT assay and cell

growth assay were performed to confirm the effects of the increased proliferative

potential due to GATA3 deficiency in BE(2)-C cells. Through western blot analyses

between GATA3 knockdown cells and the control cells, we observed that Cyclin D1

expression is highly upregulated and the E2F1 expression is dramatically

downregulated in GATA3 knockdown cells. Therefore, Cyclin D1 and E2F1 may be

the downstream targets of GATA3 in the increased proliferative status. In the

differentiation study, we demonstrated that loss of GATA3 induces glial differentiation in BE(2)-C cells, as evidenced by the upregulation of the glial cell

marker glial fibrillary acidic protein (GFAP) and the downregulation of the neuronal 6

marker SNAP25. Additionally, GATA3 overexpression promotes neuronal

differentiation in BE(2)-C cells as indicated by the downregulation of GFAP and the

upregulation of SNAP25. The regulation of the differentiation might be controlled by

the modulations between GATA3 and other sympathetic regulators, such as Phox2b and Mash1.

In summary, GATA3 deficiency induces glial differentiation in BE(2)-C cells

with increased tumorigenicity and GATA3 overexpression promotes neuronal

differentiation accompanied by a decreased proliferative potential in BE(2)-C cells.

These findings delineate the role of GATA3 in the regulation of the BE(2)-C cell tumorigenic and differentiative status. The role of GATA3 in the I-type neuroblastoma stem cell activities indicates that loss of GATA3 may contribute to the tumorigenesis, dissemination and metastasis of neuroblastoma.

LITERATURE

Tumorigenesis

Tumorigenesis is a multiple gene mutation process caused by interactions

between genetics and environment which creates progressively unrestrained

proliferation of transformed malignant cells (Kinzler and Vogelstein, 1996). Though

derived from normal cells, cancer cells acquire 6 distinguished capabilities “self-

sufficiency in growth signals, insensitivity to anti-growth signals, limitless replicative potential (immortalization), evading programmed cell death (apoptosis), sustained angiogenesis, and tissue invasion and metastasis” (Hanahan and Weinberg, 2000).

Through these acquired abilities, cancer cells undermine the balanced regulation

between cell birth and death and induce neoplastic transformation.

The basis of loss of growth regulation that gives rise to cancer is genetic mutation, which involves two broad classes of genes: proto-oncogenes and tumor suppressor genes. Functionally, the proteins encoded by both classes of genes mostly regulate the cell cycle, apoptosis and damaged DNA repair. Regulation of the

eukaryotic cell cycle is maintained through complex and delicate mechanisms. Both

positive- and negative-acting regulators tightly control the progression through each

phase of the cell cycle - G1, S, G2 and M phase - which are prime targets for cancer-

causing mutations. The restriction point is a late G1 phase checkpoint in the cell cycle.

Cells that progress through this point are irreversibly committed to enter S phase,

where DNA synthesis and replication will occur. D-type cyclins, cyclin-dependent

kinases (CDKs), and the retinoblastoma protein (pRb) are the regulators that control

the passage through the restriction point. The Cyclin D expression is induced upon

stimulation by many growth factors or mitogens. The CDK4 and CDK6 can partner

with Cyclin D to form catalytically active cyclin-CDK complexes, which promotes

the passage through the restriction point. Active Cyclin D/CDK4, Cyclin D/CDK6

and other cyclin/CDK complexes catalyze the complete phosphorylation of pRb

(Diehl, 2002). The completely phosphorylated pRb releases the active E2F transcription factors, which are important in the regulation of gene transcription required for DNA synthesis, therefore cells irreversibly enter into S phase (Weinberg,

1995). The p16 functions as a CDK inhibitor and specifically binds to CDK4 and

CDK6 thus leading to G1 arrest by inhibiting their kinase activities (Aprelikova et al.,

1995) (Figure 1). Genetic mutations occurring in this pathway or the mutations in

upstream regulators of this pathway which lead to unregulated passage from G1 to S

phase are oncogenic.

Amplification or overexpression of Cyclin D1 is important in the development

of many cancers including parathyroid adenoma, lymphoma, breast and prostate

cancers (de Boer et al., 1997; Drobnjak et al., 2000; Hsi et al., 1996; Musgrove et al.,

1994) . The p16 belongs to the family of tumor suppressor genes. The loss-of-function

of p16 occurs in several human cancers, such as melanoma, breast and lung cancers

(Belinsky et al., 1998; Freedberg et al., 2008; Milde-Langosch et al., 2001). Mutations

of both Rb alleles lead to retinoblastoma in childhood. Studies stated that only one

working allele of the Rb gene is necessary for its function, so both need to be mutated

before the cancer phenotype appears. Loss of the Rb gene function in later life

commonly combines with other gene mutations in cancers and plays a less important

role (Chau and Wang, 2003). The E2F genes are transcriptionally regulated by several

repressors, such as pRb, Swi/Snf complexes (Gunawardena et al., 2007). Increased proliferative effects of E2F proteins due to loss-of-function of repressors induce passage through the cell cycle and lead to oncogenesis. The G1 checkpoint is important for inhibiting cells with damaged DNA from entering the S phase. As “the guardian of the genome”, p53 plays an important role in this checkpoint by activating the expression of genes encoding proteins that sustain arrest in G1 and G2, promote apoptosis or exert DNA repairs. The loss-of-function mutation of p53 occurs in multiple cancers due to uncontrolled regulation of the G1 checkpoint and apoptosis

(Davidoff et al., 1991; Nozaki et al., 1999). Therefore, tumorigenesis is controlled by a complicated regulatory network inducing unbalanced control of cell birth and death which provides the foundation for anti-cancer therapies.

Figure 1. Restriction point control

Unphosphorylated Rb protein binds transcriptional factors collectively called E2F and thereby prevents E2F-mediated transcriptional activation of many genes whose products are required for DNA synthesis (e.g., DNA polymerase). The kinase activity of cyclin D-CDK4 phosphorylates Rb, thereby activating E2F; this kinase activity is inhibited by p16. Overproduction of cyclin D, a positive regulator, or loss of the negative regulators p16 and Rb, commonly occurs in human cancers. Adopted from Lodish, H. F. (2003). Molecular cell biology, 5th edn (New York, W.H. Freeman and Company), 958.

Stem Cells

The classic definition of a stem cell is that a cell has both capacities of self- renewal and differentiation into progeny cells. According to the differentiation potency, stem cells can be classified into four groups. Totipotent stem cells are fertilized oocytes (the zygotes) that have the capability to form all embryonic and extraembryonic cell types. Pluripotent stem cells are the cells arising from any of the three germ layers and are the descendants of totipotent stem cells, which have the ability to differentiate into almost all types of cells. Multipotent stem cells can differentiate into a limited range of cell lineages, based on which, most tissues are formed. Unipotent stem cells are able to produce only one cell type, such as muscle stem cells and epidermal stem cells, whereas they still keep the ability of self-renewal which distinguishes them from non-stem cells (Preston et al., 2003). There are usually several intermediate cells of increasing differentiated status, which are progenitor cells, between stem cells and their terminal progeny cells. Therefore, stem cells reflect a relatively undifferentiated cell status and are the source of committed cells, whereas they are not able to exert the special functions of progeny cells.

In addition to self-renewal and differentiation, stem cells also have other properties which may only apply to certain tissues, including the capability to go through asymmetric cell divisions; display extensive self-renewal ability; exist in a mitotically quiescent status; and regenerate all types of cells of the tissue in which they exist (Hall and Watt, 1989; Potten and Loeffler, 1990). These aspects exhibit the variety of stem cell properties besides most fundamental questions. The variety of stem cell properties reflects the difficulties in reaching a comprehensive definition of a stem cell.

Symmetric Versus Asymmetric Divisions

The self-renewal ability of a stem cell is often considered through the

asymmetric division which produces one stem cell (itself) and one committed

progenitor cell (Figure 2b). But this kind of division is not exclusive in the stem cell

self-renewal. Some stem cells can undergo symmetric divisions (Figures 2a and 2c),

allowing the pool expansion of stem cells or the production of two differentiated

daughter cells at expense of the stem cell depletion (Potten and Loeffler, 1990). The

clear examples are including the divisions in Hirudo medicinalis, Drosophila

melanogaster, Caenorhabditis elegans and the mammalian system. These two kinds of

symmetric divisions exist in one population and are regulated by the factors that

control the probability of self-renewal versus differentiative divisions. If the

probability is 0.5, the proportion of stem cells and differentiated cells remains in a

steady state in this population which can’t be distinguished from the population

behavior in Figure 2b. However, there is plenty of evidence that the size of stem cell population changes in natural environments and the probability of self-renewal versus differentiative divisions could be different from 0.5. For example, the fetal liver

hematopoietic stem cells (HSCs) double their absolute number daily during mid-

gestation phase (Morrison et al., 1995a); the long-term self-renewing HSCs increase

their absolute number more than five-fold during adult life in mice (Morrison et al.,

1996). Therefore, the symmetric divisions are fundamental for the stem cell number

regulation which is impossible in strictly asymmetric divisions. Lastly, stem cells can

exist in a quiescent status under the control of niche factors, exhibiting no self-

renewal and differentiation (Figure 2d).

Figure 2. Symmetric versus asymmetric divisions in stem cells

A stem cell can self-renew by dividing symmetrically to generate two stem cells (a), or asymmetrically to generate a stem cell and a restricted progenitor (b). A loss of either developmental potential (c) or the ability to proliferate (d) results in a failure of stem cells to self-renew. Maintenance of developmental potential means maintenance of pluripotentiality in all kinds of stem cells. Adopted from Molofsky, A. V., Pardal, R., and Morrison, S. J. (2004). Diverse mechanisms regulate stem cell self-renewal. Curr Opin Cell Biol 16, 700-707.

Self-Renewal Capacity

There is an assumed concept that the self-renewal ability of stem cells lasts for the whole life of the organism. However, there is ample evidence to support that not all stem cells possess unlimited potential of self-renewal (Abkowitz et al., 1990;

Margolis and Spradling, 1995; Morrison et al., 1995b). First, though there are adult stem cells in the organisms, some of them simply persist in a quiescent state under most conditions, as shown by the astrocyte-like stem cells in the mouse hippocampus

(Ermini et al., 2008). Second, the active stem cells during the development exist

transiently. For example, oocytes and sperms which are both derived from primordial

germ cells start to behave differently from birth, though their stem cell properties in

early gestation can’t be distinguished from females and males. Oocyte number is

stable since birth, while sperm number keeps increasing into adulthood (Donovan,

1994). Thus, no matter whether they are embryonic or adult stem cells, their self-

renewal states are different during the life, but do not last for life.

Mitotic Quiescence

Dividing slowly and rarely seems a characteristic of stem cells, but it is not

completely true, as evidenced by the mammalian intestinal crypt stem cells and the

Drosophila ovary somatic stem cells. Their dividing rate is demonstrated to be once

every 12 hours (Margolis and Spradling, 1995; Potten and Loeffler, 1990). It is generally true that adult stem cells divide slowly, such as the stem cells in the skin

(Lavker et al., 1993) and bone marrow (Morrison and Weissman, 1994), but which

can’t include all the stem cell division statuses in adults.

Regenerative Capacity

It is well known that some stem cells only display regenerative capacity

conditionally, such as in the case of tissue damage. However, it doesn’t mean that

there are no stem cells in non-regenerative tissues, like the brain (Alvarez-Buylla and

Lois, 1995; Gage et al., 1995). Reasons for the regeneration failure may derive from

lack of space, connective tissues or factors inducing the rebuilding of the cell

arrangement or promoting differentiation in the injured tissues, but not because of the

absence of pluripotent stem cells.

The regulation of stem cell self-renewal is controlled by both extrinsic and intrinsic factors and involves multiple pathways at the genetic level. Some factors are

also related to differentiation pathways, whereas others are not.

The interplay network among Nanog, Oct4, leukemia inhibitory factor (LIF)

signaling and bone morphogenetic protein (BMP) signaling is essential for the

regulation of embryonic stem (ES) cell self-renewal and the decision whether to self-

renew or differentiate (Chambers et al., 2003; Mitsui et al., 2003; Ying et al., 2003).

Oct4 and Nanog are intrinsic transcription factors that are only expressed in the inner

cell mass of blastocysts and in the germline in vivo (Palmieri et al., 1994) and are

necessary for the maintenance of pluripotency in the inner cell mass (Chambers et al.,

2003; Mitsui et al., 2003). Other transcription factors, like Foxd3, can regulate self-

renewal in both ES cells and somatic stem cells at a later developmental stage, such as

neural crest cells (Hanna et al., 2002). The underlying mechanisms for Foxd3 to

promote self-renewal are to inhibit differentiation in both pluripotent and multipotent

stem cell contexts (Dottori et al., 2001; Kos et al., 2001). In vitro studies proven that

LIF activity is dependent on BMP level in the serum. LIF promotes the self-renewal

of mouse ES cells in cultures through Stat3 activation (Niwa et al., 1998), whereas

BMP exerts its function through inhibiting the neurogenic transcription factors which

promote neuronal differentiation (Ying et al., 2003).

The Notch, Wnt and Sonic hedgehog (Shh) signaling pathways can regulate

self-renewal in many kinds of stem cells and their functions are usually context-

dependent. Notch is an important factor in diverse stem cell niches in regulating self-

renewal abilities. Notch activation promotes the neural stem cell self-renewal under some conditions (Hitoshi et al., 2002), whereas it promotes glial differentiation under

other conditions (Morrison et al., 2000; Scheer et al., 2001). The Wnt signaling pathway regulates the self-renewal of the intestinal epithelial and skin stem cells by controlling their proliferation and migration (Batlle et al., 2002; Huelsken et al., 2001).

The Shh signaling is essential for maintaining neural stem cells in multiple regions of the mammalian central nervous system (CNS) (Lai et al., 2003; Machold et al., 2003),

which is independent of its patterning effect on the nervous system.

The Mel-18, Rae-28 and Bmi-1 belong to polycomb family members, which assemble into large protein complexes to repress transcriptions by modulating

chromatin structures (Jacobs and van Lohuizen, 2002). Although Mel-18, Rae-28 and

Bmi-1 are components of the polycomb complex, they play different roles in self-

renewal regulation. The self-renewal ability of fetal HSCs increases in Mel-18

deficient mice (Antonchuk et al., 2002) whereas decreases in Rae-28 deficient mice

(Ohta et al., 2002). Bmi-1 has little effect on the embryonic development but plays an

essential role in the postnatal maintenance of hematopoietic and neural stem cells

through cell cycle regulators p16Ink4a and p19Arf (Jacobs et al., 1999a; Molofsky et al.,

2003; Park et al., 2003).

The CDK inhibitors which regulate the cell cycle progression into G1 phase

are also important in regulating self-renewal in multiple kinds of stem cells. The early

G1-phase regulator p18Ink4c and the late G1 phase regulator p21cip1 both decrease the

self-renewal in HSCs (Cheng et al., 2000b; Yuan et al., 2004). The difference is that p18Ink4c deficiency induces unlimited expansion of stem cells, whereas loss of p21cip1 leads to transient stem cell expansion (Cheng et al., 2000b). The p21cip1 and p27kip1 regulate different hematopoietic cell populations. The p21cip1 regulates the self-

renewal of HSCs, but not the proliferation of restricted hematopoietic progenitor cells,

whereas the p27kip1 doesn’t control the self-renewal of HSCs but does regulate the

proliferation of restricted hematopoietic progenitor cells (Cheng et al., 2000a; Cheng

et al., 2000b).

Cancer Stem Cell and Tumorigenesis

A cancer stem cell can be defined as “a cancer cell that has the ability to self-

renew, dividing into another malignant stem cell and a cell that gives rise to the

phenotypically diverse tumor cell population” (Bjerkvig et al., 2005). As we know,

stem cells in different tissues can self-renew and differentiate into multiple tissue

specific cell types based on their intrinsic properties (Al-Hajj and Clarke, 2004). It is also well known that most tumors are heterogeneous, comprised of diverse cell

populations which are different in the morphology, proliferative potentials, metastatic

capacities as well as tumor reconstitution abilities on transplantation.

Cancer stem cells were first identified in leukemias in1997 by John Dick and

colleagues at the University of Toronto. They harvested leukemic cells from patients

and found that only a small subset of cancer cells was capable of forming the same

cancer in immunodeficient mice (Bonnet and Dick, 1997). Cancer stem cells have

been isolated and amplified by surface makers on their membranes (Blair et al., 1997;

Jordan et al., 2000). A subpopulation of cells from human acute myeloid leukemia

(AML) expressing a CD34+/CD38- phenotype were identified as leukemia-initiating

cells by transplantation assay. Another subpopulation of cells - CD34+/CD38+ leukemic

cells couldn’t initiate leukemia in immunodeficient mice in most cases, though they

exhibit a leukemic blast phenotype (Bonnet and Dick, 1997; Sutherland et al., 1996).

On transplantation, cancer-initiating cells produce tumors composed of both new

cancer stem cells and diverse heterogeneous differentiated non-tumorigenic cells with

limited proliferative potentials. The constitution of tumor is similar to the

developmental hierarchy in the tissues from which the cancers arose. Since then,

cancer stem cells have been identified in multiple cancers, such as brain, breast, colon,

ovary, pancreas and prostate cancers (Al-Hajj et al., 2003; Li et al., 2007; Maitland and Collins, 2008; O'Brien et al., 2007; Singh et al., 2003; Zhang et al., 2008). Cancer stem cells are widely recognized as the tumor-initiating source as opposed to the

conventional cancer model.

As shown in Figure 3, in the conventional model of cancer, tumors are composed of different cell types with diverse genetic backgrounds and each type of

cells can divide and initiate tumors. However, this can’t explain why tumors which

had been shrunk to nothing after the first round treatment would soon recur. The

mysterious cell population which researchers and therapists overlooked was possibly

cancer stem cells. In the cancer stem cell model, tumors comprise diverse cell types

including cancer stem cells and only cancer stem cells can initiate tumors, but not

other cell types (Abbott, 2006). According to this model, cancer stem cells have

particular properties which distinguish them from other cell types in tumors and these

stem cells are resistant to typical therapies. They lurk for months or years, re-seed cancers and cause the recurrence usually in a more aggressive way. This theory sheds

light on the “the root of the problem (tumorigenesis)” and provides a new way for

tumor therapies - treatments to target the cancer stem cell subpopulation according to their specific properties, without damaging normal stem cells, and thus prevents

tumor recurrence.

Figure 3. Two views of cancer model

Conventional model of cancer is based on the ability of every cancer cell to form new tumors and current therapy targets the main body of tumor cells (a). But new cancer stem cell model suggests that only a subset of tumor cells which possess the stem cell properties can form new tumor. If target this specific cell group, the tumor recurrance will be prevented. Adopted from Abbott, A. (2006). Cancer: the root of the problem. Nature 442, 742-743.

The exact origin of cancer stem cells in tumors is not quite clear yet. They could derive from normal stem cells that have accumulated oncogenic mutations over time and are malignantly transformed. This idea is proven by the evidence that many kinds of cancer stem cells express the cell surface markers which are also found in normal stem cells (Fillmore and Kuperwasser, 2007; Reiter et al., 1998). But normal stem cells might not be the only origin of cancer stem cells. The origin of tumor cells could be a more differentiated cell which acquires the continuous self-renewal ability and gains stem cell properties (Cozzio et al., 2003). Krivtsov and colleagues found a leukemia-associated fusion gene that functions to turn non-stem cells into cells acquiring stem cell behaviors and thus induce tumor formation (Krivtsov et al., 2006).

This gene is produced by the ‘mixed lineage leukemia’ gene (MLL) fusing with AF9 gene and is closely related to the development of AML (Daser and Rabbitts, 2004).

Myeloid progenitor cells are derived from HSCs and differentiate into further specialized progenies, so they don’t possess self-renewal ability and are normally destined to produce mature white blood cells or myeloid cells (Akashi et al., 2000).

The MLL-AF9 fusion gene induces myeloid progenitor cells to aberrantly express a specific set of ‘stem cell genes’ and turns them into cancer stem cells capable of initiating, maintaining and propagating the leukemia (Krivtsov et al., 2006).

It has been proven that several pathways that regulate normal stem cell self- renewal are shared by neoplastic proliferation, but in a deregulated mutated style, such as Notch, Wnt, Shh, Pten, Bmi-1 signaling pathways. These signaling pathways have been demonstrated to regulate self-renewal in somatic stem cells and also contribute to the carcinogenesis in the same tissue when these pathways are

deregulated ( Table 1) (Di Cristofano and Pandolfi, 2000; Lessard and Sauvageau,

2003; Pear et al., 1996; Polakis, 1999; Wetmore, 2003).

Notch activation has been shown to consistently increase the amount of

primitive progenitor cells in the HSC culture, which suggests that Notch signaling

upregulates the HSC self-renewal or maintains the multipotentiality of hematopoietic

progenitor cells (Karanu et al., 2000; Varnum-Finney et al., 2000). Moreover, the constitutively activated Notch pathway is linked to a subset of acute T-cell

lymphoblastic leukemias (Pear et al., 1996). β-catenin is the downstream molecule of

Wnt signaling and located in the cytoplasm. Constitutive activation of β-catenin

promotes the self-renewal of CNS and keratinocyte stem cells and induces CNS and

skin tumors (Gat et al., 1998; Zurawel et al., 1998). In the intestinal epithelial stem

cell niche, Wnt signaling regulates stem cell self-renewal primarily through two

downstream pathways. One is EPH-family adhesion molecules (Batlle et al., 2002)

which control the migration out of niche (crypt) and induce differentiation. The other

is c-Myc (van de Wetering et al., 2002) which promotes proliferation. Wnt signaling

activates the same downstream molecules in colorectal cancers (van de Wetering et al.,

2002) and causes the hyperproliferation of crypt progenitor cells, producing polyps

comprising multiple lineage progenies (Moser et al., 1992; Powell et al., 1992). Wnt

signaling is also implicated in hematopoietic malignancies based on its property to

promote self-renewal in HSCs (Chung et al., 2002; Qiang et al., 2003). Shh signaling

is essential for the cerebellum development. Shh proteins are secreted by Purkinje

cells and support the proliferation of granule-cell precursors at a high rate while

inhibit their terminal differentiation (Kenney and Rowitch, 2000; Wechsler-Reya and

Scott, 2001). In the normal development, however, most granule-cell precursors cease

to proliferate around postnatal 14 days even in the continuous presence of Shh, which

is possibly due to the accumulation of p27 (Miyazawa et al., 2000). Medulloblastoma

originates from granule-cell precursors and is characterized by neural progenitor

expansion, (Pietsch et al., 1997; Wechsler-Reya and Scott, 2001) which might be

caused by Shh signaling to maintain the unlimited proliferative capacity in progenitor

cells through suppression of pRb (Marino et al., 2000).

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN)

tumor suppressor gene is one of the most mutated genes in glioblastoma (Li et al.,

1998). PTEN deficiency induces PIP3 accumulation and activates signaling pathways

that regulate cell size, cell migration, cell death, cell proliferation and differentiation which are closely related to tumorigenesis (Stiles et al., 2004). PTEN deletion leads to

increased capacity to the neural stem/progenitor cell self-renewal through promoting

exit from the G0/G1 phase into S phase. So PTEN might play an essential role in

brain tumorigenesis (Groszer et al., 2006). Recently, Yilmaz et al. found that in the

hematopoietic system, PTEN deletion leads to generation of leukemic stem cells but

depletion of normal HSCs, which implies that the underlying mechanisms

maintaining pools of leukemic stem cells and normal HSCs are distinct (Yilmaz et al.,

2006). This distinction may realize the goal that therapies are designed to combat

leukemia targeting the PTEN pathway without affecting normal stem cell pools. Bmi-

1 is one member of the polycomb family which works as a transcriptional repressor

through chromatin remodeling. Bmi-1 is essential for self-renewal of HSCs as well as

for that of leukemic stem cells partly through two CDK inhibitors, p16Ink4a and p19Arf

(Jacobs et al., 1999a; Jacobs et al., 1999b). Bmi-1 is also required for self-renewal of stem cells in the peripherin nervous system (PNS) and CNS but not for their survival

or differentiation (Molofsky et al., 2003). This relates Bmi-1 to the generation of multiple brain tumors, such as medulloblastoma (Leung et al., 2004), glioblastoma

(Godlewski et al., 2008) and PNS tumors, such as neuroblastoma (Cui et al., 2007).

Bmi-1 overexpression was found in the majority of human medulloblastomas

analyzed, which is correlated to the overexpression of PTCH1, a reliable indicator of

Shh pathway activation (Leung et al., 2004). Bmi-1 is also demonstrated as an

important regulator in balancing differentiation and clonogenic self-renewal in I-type

neuroblastoma cells, which are considered as a population of malignant neural crest stem cells (Cui et al., 2006).

Various stem cells are characterized by expressing drug-resistance proteins,

such as MDR1 and ABC transporters (Chaudhary and Roninson, 1991; Zhou et al.,

2001), which might be the reasons for less sensitivity to chemotherapy and apoptosis

induction (Johnstone et al., 1999; Pallis and Russell, 2000). If cancer stem cells also

express these proteins at a higher level than do differentiated cancer cells,

chemotherapy may not be able to kill these resistant cells. Chemotherapy that kills

more differentiated cancer cells with limited proliferative potential would lead to the

shrinkage of tumor. But the surviving cancer stem cells still will induce tumorigenesis.

Therefore, identification of agents that particularly target cancer stem cells is an

essential way of preventing tumor recurrence. The gene expression profile of cancer

stem cells, differentiated cancer cells with limited proliferative potential and stem

cells is indicated to be different in a given patient (Al-Hajj et al., 2003; Bonnet and

Dick, 1997; Lapidot et al., 1994). So comparison of subpopulation RNA profiles in

the same tumor, rather than using the whole tumor’s RNA will help to identify

therapeutic targets that are preferentially expressed in cancer stem cells. The stem

cells and cancer stem cells tend to depend on signal pathways at different levels to

maintain self-renewal or neoplastic proliferation because the extent of activation of a

certain pathway is dependent on the mitotic activity of cells, the amount of

regenerative activity in the tissue and the stage of development. The status of stem

cells and cancer stem cells is usually different in these aspects, which provides the

possibility of targeting cancer stem cells without harming normal stem cells. If cancer stem cells can be specifically eliminated, the possibility of cancer recurrence will be dramatically decreased.

Neural Crest Cells and Sympathetic Development

Neural crest stem cells (NCSCs) are a transit population of cells from which

diverse derivatives originate (LaBonne and Bronner-Fraser, 1998). Trunk NCSCs that

migrate dorsally develop into pigment-synthesizing melanocytes and dorsal root

ganglia containing sensory neurons. Ventrally migrating NCSCs differentiate into

sympathetic ganglia, adrenal medulla, vertebral cartilage and nerve clusters around

aorta. In rodents, migrating and post-migratory NCSCs can be identified by their

expression of the markers Sox10, FoxD3, nestin and p75, the low affinity nerve

growth factor receptor (NGFR). Sox10, a member of the SRY related HMG box gene

family of transcription factors, is essential for maintaining the stem cell state of

NCSCs (Kim et al., 2003). More recent studies revealed that Bmi-1, a member of the

Polycomb Group family of transcription repressors, is required for the self-renewing

proliferation of NCSCs. Bmi-1-deficient mice display progressive loss of NCSCs,

and Bmi-1-/- NCSCs show reduced ability to self-renew in cultures (Molofsky et al.,

2005; Molofsky et al., 2003).

Sympathetic ganglia are derived from ventrally migrating NCSCs arising from

the trunk region of the neural tube. Two columns of sympathetic ganglion primordia are formed along the dorsa aorta during the early embryonic development. Through a series of differentiation processes, NCSCs give rise to noradrenergic neuroblasts and glial progenitors that coalesce to form definitive sympathetic ganglia. In rodent superior cervical ganglia (SCG), neurogenesis peaks on embryonic day 14.5 (E14.5) and is completed by E17.5 (Hall and Landis, 1991), and gliogenesis peaks around

E16.5-E18.5 and continues at a lower level during the first 3 weeks after birth (Hall and Landis, 1992; Shi et al., 2008).

Sympathetic neurogenesis has been widely used as a model for the study of

the neuronal development. A commonly used marker for monitoring sympathetic

neurogenesis is tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis

of noradrenaline that characterizes the noradrenergic trait of sympathetic neurons.

BMPs, produced by and secreted from the dorsal aorta, serve as an extracellular signal

essential for sympathetic neuronal differentiation. Addition of BMP2, 4, or 7 or

overexpression of a constitutively active BMP receptor dramatically increases the

number of TH-expressing cells in NCSC cultures (Reissmann et al., 1996; Varley et al., 1998). More importantly, implantation of beads soaked in the BMP antagonist

Noggin in the vicinity of the dorsal aorta prevents the expression of noradrenergic and

neuronal markers in NCSCs, demonstrating an essential role of BMPs in the

generation of sympathetic neurons in vivo (Schneider et al., 1999). At molecular

levels, the binding of BMPs to their receptors on NCSCs initiates a cascade of events

that eventually leads to the expression of a group of transcription factors that controls

the differentiation of sympathetic neurons (Figure 4). This group of transcription

factors includes Mash1, Phox2a, Phox2b, dHand and GATA3.

Figure 4. Sympathetic neuronal development

The specification is initiated by BMPs and transcription factors Mash1, Phox2b, dHand and GATA3, which in turn control the expression of noradrenergic (TH and DBH) and generic neuronal (NF160 and SCG10) properties. Adopted from Goridis, C., and Rohrer, H. (2002). Specification of catecholaminergic and serotonergic neurons. Nat Rev Neurosci 3, 531-541.

In NCSC cultures, addition of BMP2 can induce the expression of Mash1 and

Phox2a (Lo et al., 1998). Mash1 is a helix-loop-helix transcription factor and is

transiently expressed around E10.5 during mouse sympathetic neurogenesis

(Guillemot and Joyner, 1993). In Mash1-/- mouse embryos, NCSCs assemble normally

at the dorsal aorta, but most of them fail to express pan-neuronal markers or

noradrenaline biosynthetic enzymes TH and dopamine β-hydroxylase (DBH)

(Guillemot et al., 1993). Mash1 appears to function upstream of Phox2a because the

constitutive expression of Mash1 in NCSCs induces expression of Phox2a and

neuronal differentiation (Lo et al., 1998). Also in Mash1-/- embryos, expression of

Phox2a in sympathetic ganglia is strongly reduced, but the expression of Phox2b,

dHand, neurofilaments and neuron-specific tubulin is not affected (Guillemot et al.,

1993; Lo et al., 1998). BMPs can also induce the expression of the homeodomain

protein Phox2b, independently of Mash1. Phox2b is expressed earlier than Phox2a

and also plays a role in regulation of Phox2a expression (Pattyn et al., 1997; Pattyn et

al., 1999; Schneider et al., 1999). Mice deficient in Phox2b display more severe

defects in sympathetic neurogenesis. Although in Phox2b-null embryos, NCSCs can

assemble at the dorsal aorta on E10.5, these cells fail to express dHand,

neurofilaments and neuron-specific tubulin, indicating a block in generic neurogenesis which is not affected by Mash1 mutation (Pattyn et al., 1999). In addition, Mash1 expression is downregulated in Phox2b-/- mouse embryos (Pattyn et al., 1999). The

dHand basic helix-loop-helix transcription factor plays an essential role late in the sympathetic neurogenesis. The expression of dHand depends on Phox2b but not on

Mash1 (Howard et al., 2000; Morikawa et al., 2005). Forced expression of dHand induces the expression of noradrenergic biosynthetic enzymes and pan-neuronal markers in NCSCs (Howard et al., 2000; Morikawa et al., 2005). GATA3 is a zinc 31

finger transcription factor. In GATA3 mutant embryos, sympathetic ganglia express

Phox2a and Phox2b, as well as pan-neuronal markers. However, the neurons fail to

express TH and DBH, indicating a block in the maturation of sympathetic neurons

(Lim et al., 2000).

It is generally thought that gliogenesis represents a default pathway of NCSCs

differentiation. Sox10 is the only gene known to be essential for the generation of

glial cells from truck NCSCs. Sox10 expression persists in sympathetic glial

progenitors and fully differentiated glial cells (Jessen and Mirsky, 2005). One function of Sox10 in gliogenesis might be to preserve the ability of NCSCs and/or glial progenitors to respond to neuregulin 1 (NRG1) by maintaining the expression of

ErbB3, the NRG1 receptor (Britsch et al., 2001). NRG1 and Notch ligands are two extracellular factors with the gliogenic promoting function. Both appear to function

indirectly inhibiting neurogenesis (Jessen and Mirsky, 2005). Several markers are

available for the identification of sympathetic glial cells and their progenitors. The

mostly commonly used markers are glial fibrillary acidic protein (GFAP) and S100

for differentiated glial cells, and brain lipid binding protein (BLBP) for both

progenitor and differentiated glial cells.

Neuroblastoma

Neuroblastoma is a common childhood solid cancer and originated from

NCSCs or sympathetic precursor cells (Brodeur, 2003). 90% of children with this

disease are diagnosed before the age of 6 years (Schwab et al., 2003). Neuroblastoma

can either regress spontaneously, usually in infants, or mature into a benign

ganglioneuroma by undergoing apoptosis and/or differentiation, whereas in children

over 1 year old, the tumors are mostly aggressive and fatal, and their overall prognosis

has been poor (Brodeur, 2003; Nakagawara, 1998). Neuroblastoma has diverse

clinical features because of its variable origin sites, inclination to metastasis, hormone

secretion and manifestation as a paraneoplastic syndrome (Schwab et al., 2003). The enigmatic clinical behavior of neuroblastoma is also based on its heterogeneous cellular composition, genetic mutations and biological pathways.

Three distinct types of cell lines were established from human neuroblastomas

based on their morphologies, biochemical properties and growth patterns. They are N

for neuroblastic, S for non-neuronal, substrate-adherent and I for intermediate cell

types (Ross et al., 2003). N-type cells have immature neuroblastic features with small

and rounded bodies, a high nuclear to cytoplasmic ratio and short neurites. They

attach better to other cells than to substrate and form cell aggregates in culture. They

express neurofilaments, specific noradrenergic neuronal enzymes, such as TH and

DBH, and cell surface receptors in developing neuroblasts, like the norepinephrine

uptake transporter and the low affinity nerve growth factor receptor (Ross et al., 2002).

S-type cells show characteristics opposite to those of N-type cells. They are large and

flattened cells with abundant cytoplasm. They may have long filopodia but no

neurites. These cells adhere tightly to substrate, grow as a monolayer in culture and

present contact inhibition of growth. S-type cells express no neuronal markers and

have phenotypes of Schwann/glial cells, melanocytes and smooth muscle cells

(Ambros and Ambros, 1995; Sugimoto et al., 1991). They express glial cell markers

like vimentin and GFAP and other proteins such as the epidermal growth factor

receptor, melanocyte enzyme tyrosinase and alpha-smooth muscle actin. I-type cells

exhibit an intermediate status between N-type and S-type cells. They attach equally

well to both the substrate and other cells. They may or may not have neurites and

form multilayers with focal aggregates in cultures. They have nuclei similar to N-type

cells and more cytoplasm like S-type cells. I-type cells express both N- and S- type

cell markers and stem cell markers, such as CD133 and c-kit (Walton et al., 2004). It

has been proposed that I-type cells may represent a population of neuroblastoma stem

cells based on its differentiation and clonogenic ability. I-type cells can differentiate

into both N-type and S-type cells under different conditions (Acosta et al., 2009). N-

type cells can be induced to neuronal or neuroendocrine cells (Ross et al., 2002)

whereas S-type cells can further differentiate into Schwann/glial cells, melanocytes

and smooth muscle cells (Slack et al., 1992; Sugimoto et al., 2000). I-type cells have

the greatest clonogenic activity in soft agar and tumorigenic potential in

immunodeficient mice. S-type cells are non-tumorigenic and N-type cells exhibit

tumorigenic ability depending on particular cell lines (Spengler et al., 1997).

Pathological analyses of primary neuroblastoma samples exhibit that tumors are

comprised of neuroblast-like cells and stroma cells (Mora et al., 2001). I-type cells are hidden in these two kinds of cells morphologically and can be identified by stem cell markers or combination with tumor markers (Kristiansen et al., 2004; Poncet et al.,

1996; Reynolds, 2004). It has been demonstrated that I-type like cells are detectable in tumors of all stages and related a high possibility in tumor progression (Ross et al.,

2003). Therefore, I-type cells in primary tumors may present a subpopulation of cancer stem cells that are the origin of tumorigenesis.

Neuroblastoma cells, like cells of many other tumor types, suffer from extensive, non-random genetic mutations at multiple genetic loci that provide genetic

basis for their diverse clinical behaviors. The most common genetic alterations in

neuroblastoma are MYCN amplification, gain of chromosome 17q distal region, and

loss of heterozygosity (LOH) for chromosomes 1p36 and 11q23 (van Noesel and

Versteeg, 2004). The relevant genes that are affected by 17q gain, 1p36 or 11q23

LOH in neuroblasoma remain to be identified. MYCN amplification is found in 20-

25% of all neuroblastomas and is related to a poor prognosis (Canete et al., 2009).

The amplification values are generally around 50 to 100 fold in tumors, but may also range from 10 to 500 fold (Schwab et al., 2003). MYCN has tumorigenic potential as

evidenced by co-operating to transform primary cells, converting established cell lines to possess tumorigenic capacities and initiating tumorigenicity in genetically engineered mice (Schwab et al., 1985; Small et al., 1987; Weiss et al., 1997). The

mechanisms underlying MYCN malignant properties are its ability to promote

proliferation (Lutz et al., 1996) and prime cells for apoptosis by sensitizing them to an array of damages. The evasion of MYCN primed apoptosis is required for tumor aggression in neuroblasts with MYCN amplification (Hogarty, 2003). The evasion is likely to result from co-operating mutations that activate survival pathways or inhibit apoptosis pathways. In survival pathways, constitutive neurotrophin signaling may

suppress apoptosis in neuroblasts and the signaling pathway may involve

neurotrophins and their receptors- TrkA, TrkB and TrkC (Evangelopoulos et al.,

2004). The mutations from both mitochondrial and death receptor apoptosis pathways

contribute to evasion from MYCN primed apoptosis, such as Bcl2 overexpression and

Caspase 8 inactivation (Schmitt and Lowe, 2001; Teitz et al., 2000).

Neuroblastoma is one of few cancers originating from the embryonic

development, which implies that this tumor may be initiated by transformed

developing NCSCs or malignant sympathetic precursor cells that obtain the stem cell

properties through mutations. This neuroblastoma stem cell model can adequately

interpret the heterogeneous characteristic of human neuroblastoma tumors, based on

the self-renewal and differentiation ability of malignant NCSCs or sympathetic

precursor cells. I-type neuroblastoma cell lines have been identified as cancer stem

cells based on their special phenotypic characterization, differentiation potential and

malignant potential (Ross and Spengler, 2007). The existence of stem-like cells in

neuroblastoma tumors was demonstrated using dye exclusion with fluorescence

activated cell sorting (FACS) analysis (Hirschmann-Jax et al., 2004) and coexpression of proteins from different neural crest cell lineages (Walton et al., 2004). It was observed that in high mortality rate neuroblastomas, usually in children over 1 year old, many tumor stem cells exist and differentiated tumor cells are less than those of favorable prognosis tumors (Cassady, 1984). Since neuroblastoma cancer stem

cells play an essential role in the tumor initiation, it is necessary to investigate the

regulatory mechanisms underlying self-renewal and differentiation capacities of

neuroblastoma stem cells, which provide the foundation for appropriate future

therapies in neuroblastomas. The research in our lab indicated that Bmi-1, a NCSC

self-renewal regulator, controls the delicate balance of clonogenic self-renewal and

differentiation of I-type neuroblastoma stem cells in a concentration-dependent

manner (Cui et al., 2006). My research demonstrated that GATA3, a transcription

factor in normal sympathetic neurogenesis, also plays an important role in the

regulation of I-type neuroblastoma cell activities.

GATA3, a Member of GATA Family Transcription Factors

The GATA family is a group of evolutionary conserved transcriptional factors that have been shown to play crucial roles in the development, including regulation of differentiation, proliferation, cell-fate specification and cell movement, et al. These factors have been found in all eukaryotic organisms from fungi to plants and from

invertebrates to vertebrates (Lowry and Atchley, 2000). In vertebrates, the GATA family contains 6 six members (named GATA1 to 6) which belong to zinc finger transcription factors. They bind to the common consensus DNA sequence

(A/T)GATA(A/G) from which the family name originated (Patient and McGhee,

2002).

The vertebrate GATA family proteins contain a conserved DNA-binding

domain comprised of two multifunctional zinc fingers. The C-terminal zinc finger is

responsible for site-specific recognition and DNA-binding to the core GATA motif,

whereas the N-terminal zinc finger contributes to the specificity and stability of the

DNA-binding (Figure 5) (Martin and Orkin, 1990; Omichinski et al., 1993; Yang and

Evans, 1992). Since the GATA family members share a highly conserved DNA-

binding domain, they all show similar DNA-binding properties (Ko and Engel, 1993;

Merika and Orkin, 1993). The specificity of GATA is partly governed through

protein-protein interaction with other transcription factors (Charron and Nemer, 1999;

Molkentin, 2000). There is an extensive list of ubiquitously expressed or cell-

restricted factors that are identified as cooperating with GATA factors to control

tissue specific transcription in the hematopoietic system, the heart, the pituitary, the

adrenal glands, the gonads and many other tissues (Durocher et al., 1997; Gordon et

al., 1997; Jimenez et al., 2003; Merika and Orkin, 1995; Morin et al., 2000). The

multitype zinc finger proteins named Friend of GATA1 (FOG1) and Friend of

GATA2 (FOG2) are the most notable GATA-interacting factors, which were

originally identified as GATA-specific cofactors interacting with N-terminal zinc

fingers of the GATA family members (Holmes et al., 1999; Tsang et al., 1997). FOG1

is highly coexpressed with GATA1 in hematopoietic cell lineages (Tsang et al., 1997).

FOG2, with GATA4, is highly expressed in the heart, brain, and gonads (Laitinen et

al., 2000; Lu et al., 1999). FOG proteins act as either enhancers or repressors of

GATA transcription activity in different cell contexts. Although FOG proteins don’t

bind to DNA directly, they still play crucial roles in the vertebrate development just as

GATA factors (Fox et al., 1999; Robert et al., 2002; Svensson et al., 2000a). FOG1 knockdown results in the failure in erythroid and megakaryocytic differentiation

(Tsang et al., 1998), while loss of FOG2 leads to defects in the heart morphogenesis

and the coronary vascular development (Crispino et al., 2001; Svensson et al., 2000b;

Tevosian et al., 2000), as well as the impaired gonad development (Tevosian et al.,

2002). The activity of GATA factors is also regulated by posttranslational modifications such as sumoylation, acetylation and phosphorylation. The effect of these modifications includes enhanced transcriptional activities due to changes in the nuclear localization, DNA-binding, protein stability, and cofactor recruitment of

GATA family factors (Viger et al., 2008).

Figure 5. Structures and homology of the vertebrate GATA proteins

All GATA factors share a similar zinc finger DNA-binding domain, a feature that defines this family of transcription factors. The zinc finger region is also involved in protein interactions with cofactors and/or other transcriptional partners. Transactivation domains are located in the N-terminal and C-terminal regions. The percent homology among the different GATA proteins (as deduced from the mouse sequences) in the N-terminal, C-terminal and zinc finger domains is indicated. Adopted from Viger, R. S., Guittot, S. M., Anttonen, M., Wilson, D. B., and Heikinheimo, M. (2008). Role of the GATA family of transcription factors in endocrine development, function, and disease. Mol Endocrinol 22, 781-798.

The six GATA family members can be divided into two subgroups based on

their spatial and temporal patterns (Figure 5). GATA1/2/3 are expressed in

hematopoietic cell lineages and play an essential role in the proliferation of HSCs, the development of T lymphocytes and the differentiation of erythrocytes and

megakaryocytes (Weiss and Orkin, 1995). However, their expression is not limited in

the hematopoietic system, but is also shown in the nervous system, kidney, inner ear,

et al., and is important in the development of these organs (George et al., 1994;

Labastie et al., 1995; Lillevali et al., 2004; Nardelli et al., 1999). GATA4/5/6 are

mainly expressed in the tissues originated from the mesoderm and endoderm, such as

the heart, gut and gonads (Molkentin, 2000). The mutation and expression modulation

of GATA family factors can cause human diseases. GATA1 mutation results in

dyserythropoietic anemia and thrombocytopenia (Nichols et al., 2000).

Hypoparathyroidism, deafness, and renal dysplasia -HDR syndrome- is caused by a

GATA3 mutation (Van Esch et al., 2000). A GATA4 mutation induces congenital

heart defects (Garg et al., 2003).

GATA3 plays an essential role in the development of many tissues in

vertebrates, including Th2 cell differentiation, the development of the kidney, the

inner ear, embryonic and pubertal mammary glands, adipocytes and neurons, et al.

GATA3 is essential for differentiation of T helper type 2 (Th2) cells in the

immune system. T helper type 2 (Th2) cells secrete IL-4, IL-5, IL-10, and IL-13 and

mediate allergic and asthmatic diseases. GATA3 is required for optimal Th2 cytokine

production in vitro and in vivo and must be sustained to maintain the Th2 phenotype

(Pai et al., 2004). T cell receptor-mediated activation of the Ras-ERK-MAPK cascade

stabilizes GATA3 protein in developing Th2 cells through inhibiting the ubiquitin-

proteasome pathway and facilitates GATA3-mediated chromatin remodeling at Th2

cytokine gene loci leading to successful Th2 cell differentiation (Yamashita et al.,

2005). The differentiation of T helper progenitors to Th1 or Th2 effector cells requires

the action of two opposing transcription factors, T-bet and GATA3. T-bet is essential

for the development of Th1 cells, comparable to the similar role of GATA3 in the Th2

development. T-bet represses Th2 lineage commitment through tyrosine kinase-

mediated interaction between the two transcription factors that interferes with the

binding of GATA3 to its target DNA (Hwang et al., 2005), while GATA3 expression

in developing Th1 cells suppresses the Th1 development through downregulation of

Stat4 (Usui et al., 2003).

Human GATA3 haploinsufficiency leads to HDR (hypoparathyroidism,

deafness and renal dysplasia) syndrome, demonstrating the essential role of GATA3

in the kidney and inner ear development. Nephric duct-specific inactivation of

GATA3 leads to massive ectopic ureter budding which results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. GATA3 inactivation also causes premature nephric duct cell differentiation (Grote et al., 2008).

In the inner ear development, GATA3 deficiency leads to severe and unique abnormalities during otic placode invagination. Loss of GATA3 also alters the expression of several cell adhesion mediating genes, which suggests that GATA3 controls adhesion and morphogenetic movements in the early otic epithelium

(Lillevali et al., 2006). Hearing loss following GATA3 haploinsufficiency is

peripheral in origin and is caused by a cochlear disorder (van der Wees et al., 2004).

GATA3 plays essential roles in the development of mammary glands in both embryos and adults. In embryos, loss of GATA3 leads to an expansion of luminal progenitors and a concomitant block in differentiation. Introduction of GATA3 into a stem cell-enriched population induces maturation along the alveolar luminal lineage

(Asselin-Labat et al., 2007). In pubertal mice, GATA3 is found in the luminal cells of

mammary ducts and the body cells of terminal end buds (TEBs). Upon conditional

deletion of GATA3, mice exhibit severe defects in the mammary development due to

failure in TEB formation. After acute GATA3 loss, adult mice display

undifferentiated luminal cell expansion with basement-membrane detachment, which

leads to caspase-mediated cell death in the long term (Kouros-Mehr et al., 2006).

These studies provide evidence for GATA3 as a critical regulator of luminal

differentiation.

GATA3 is crucially involved in epidermal and hair follicle differentiation

(Kaufman et al., 2003). A conditional GATA3-/- mouse in which GATA3 is

specifically deleted in the epidermis and hair follicles shows aberrant postnatal

growth and development, delayed hair growth and maintenance, abnormal hair follicle

organization and irregular pigmentation. After the first hair cycle, the germinative

layer surrounding the dermal papilla is not restored, whereas proliferation is

pronounced in basal epidermal cells (Kurek et al., 2007).

GATA2 and GATA3 are expressed in adipocyte precursors and control the

preadipocyte-to-adipocyte transition. Constitutive expression of both GATA2 and

GATA3 suppresses the adipocyte differentiation primarily with two mechanisms. One

is through direct binding to the peroxisome proliferator-activated receptor gamma

(PPARgamma) promoter and suppression of its basal activity (Tontonoz et al., 1994).

The other is through forming protein complexes with CCAAT/enhancer binding

proteins alpha (C/EBPalpha) and C/EBPbeta, members of a family of transcription

factors that are integral to adipogenesis (Tong et al., 2005).

GATA3 is an important transcription factor in the development of both CNS

and PNS. In the CNS, for example, GATA3 is expressed in the developing

serotonergic neurons of the caudal raphe nuclei. Absence of GATA3 affects the

cytoarchitecture of serotonergic neurons and knockout mice show a serious defect in

their locomotor performance on a rotating rod (van Doorninck et al., 1999). GATA3

and Stem Cell Leukemia (SCL) gene, a basic helix-loop-helix (bHLH) transcription factor gene, are coexpressed in V2 interneurons of the spinal cord and regulate their

differentiation (Smith et al., 2002). In the PNS, GATA3 is essential for the

development of sympathetic nervous system. GATA3 knockout mouse embryos show

reduced synthesis of tyrosine hydroxylase (TH) (Lim et al., 2000).

In addition to the above functions, GATA3 is also involved in the early stage

of the embryonic development, such as the regulation of trophoblast-specific gene

expression and placental function (Ma et al., 1997).

GATA3 and Sympathetic Neuronal Development

In 1995, Pandolfi et al. found that GATA3-/- embryos die by E11 and display

massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross fetal liver hematopoietic aberrations (Pandolfi et al., 1995).

In 2000, Lim et al. reported that this GATA3-induced embryonic lethality can be partially rescued by a catecholamine supplement, which suggests noradrenaline 43

deficiency in the sympathetic nervous system might cause embryonic lethality in

these mutants (Lim et al., 2000). They discovered that null mutation of GATA3 leads

to dramatic downregulation of TH and DBH mRNA, whereas the expression of

Phox2a, Phox2b, neurofilaments, p75 NGFR and β-tubulin III in the sympathetic

nervous system is not affected. Due to the important roles of GATA3 in the

development of the cephalic neural crest cells, metanephric kidney, thymus and heart, pharmacologically rescued GATA3-/- embryos exhibit severely hypoplastic mandibles, serious metanephric and thymic hypoplasia, loosely dispersed myocardium and severely hypoplastic ventricular walls as well as blood congestion. These defects may constitute other reasons for embryonic lethality.

The generation of sympathetic neurons from NCSCs is induced by the extrinsic

signal BMPs, which initiates the expression of a network of transcription factors that,

in turn, control sympathetic neuronal differentiation (Goridis and Rohrer, 2002).

Mash1 is the first transcription factor identified to be essential for sympathetic

neurogenesis (Guillemot et al., 1993). Phox2b is another essential transcriptional

factor which is required for Mash1 expression and independent of Mash1 (Pattyn et

al., 1999). Mash1 and Phox2b are genetically upstream of Phox2a and dHand (Flora

et al., 2001; Stanke et al., 2004). Forced expression of Phox2a and dHand induces the expression of the upstream genes Mash1 and Phox2b, so the interactions are reciprocal (Goridis and Rohrer, 2002). Phox2a, Phox2b and dHand bind directly to the promoters of the TH or DBH gene and activate their transcription (Rychlik et al.,

2003; Xu et al., 2003). These previous researches propose the questions: Is GATA3

one of the transcription factors in this network? What is the relationship between

GATA3 and other factors? Does GATA3 bind directly to the TH or DBH promoter to

induce their expression?

In 2004, Tsarovina et al. were trying to answer these questions in their research.

They used chicks as the model to investigate the essential role of GATA factors in the

sympathetic development (Tsarovina et al., 2004). They found that GATA2 rather

than GATA3 is involved in chick noradrenergic differentiation, whereas GATA2 and

GATA3 are expressed at the equivalent levels in mouse sympathetic ganglia. It was

found that temporally, GATA2 is expressed after the onset of Cash1, Phox2a, Phox2b and dHand expression, but before the noradrenergic marker genes TH and DBH.

GATA2 expression is also dependent on BMP signaling as evidenced by BMP loss-

of-function experiments. These facts suggest that GATA2/3 might also be involved

in the network of BMP induced transcription factors. Further experiments proved that the expression level of GATA2 and TH is upregulated in gain-of-function studies of

Phox2b, Mash1 and Phox2a. In Phox2b knockout mice, the expression of GATA2 and GATA3 in E10.5 sympathetic ganglion primordia can’t be detected. Therefore,

GATA2/3 may act downstream of Phox2b in the BMP-induced network. In GATA2 knockout chick embryos, TH expression is strongly reduced by 50%, which is consistent with the effects of GATA3 knockout on the noradrenergic gene expression in mice shown in Lim et al.’s research in 2000. They further found a reduction in sympathetic ganglion size in GATA2 knockout chick embryos, reflected by a smaller area of Phox2b- and SCG10-expressing cells, which suggests a more general role in the sympathetic development than reported for mouse GATA3. In order to find more facts, Tsarovina et al. re-investigated the effects in the GATA3 knockout mouse model. On E10.5, the sympathetic ganglion primordia shows normal size in GATA3-

/- mice, as demonstrated by the expression of transcriptional control genes (Mash1,

Phox2b, Phox2a, dHand), noradrenergic markers (DBH, TH), the GFL receptor signaling subunit (Ret) and generic neuronal markers (TUJ1). The only differences

compared with the control ganglia are lack of GATA2 expression and substantially

lower TH expression levels, whereas DBH expression is intact. On E11.5, the size of sympathetic ganglia is dramatically reduced in GATA3-/- mice due to a strong

increase in the number of apoptotic cells. On E13.5, only rudiments of ganglia are

left.

In 2006, Moriguchi et al. confirmed that there are increased apoptosis and

significant impairment of sympathoadrenal differentiation in adrenal chromaffin cells

and sympathetic neurons in GATA3 knockout mouse embryos. Through mRNA

analyses of purified chromaffin cells from GATA3 mutants, the relative expression

levels of Mash1, dHand and Phox2b (postulated upstream regulators of GATA3) as

well as downstream products TH and DBH, are markedly downregulated. The

researchers made another transgenic mouse model that specifically expresses GATA3

in sympathoadrenal lineages under the human DBH promoter. This lineage specific

restoration of GATA3 recovers sympathoadrenal function almost to the normal level

and phenotypically restores downstream, as well as the putative upstream genes,

which provides solid evidence that sympathoadrenal differentiation is controlled by

mutually reinforcing feedback transcriptional interactions between GATA3, Mash1,

dHand and Phox2b. Although full function restoration in the sympathoadrenal system,

transgenic-rescued mutant embryos still exhibit hypoplastic mandibles and glandular

aplasia (the parathyroid glands and thymuses) as well as inner ear and kidney deficiencies, which suggests the significance of GATA3 in the development of these

organs. However, the hearts of transgenic-rescued mutant embryos have normal

cardiac thickness that is indistinguishable from wild-type littermates, which implies

that the heart development is dependent more on noradrenalin than GATA3 itself

(Moriguchi et al., 2006).

In 2006, Hong et al. found that the mechanism underlying GATA3 regulation

of TH expression is not through directly binding to the TH promoter as other

transcription factors like Phox2a, Phox2b and dHand. GATA3 interplays with transcription factor CREB by protein-protein interaction. CREB binds to a cAMP response element (CRE) in the TH promoter area and promotes the TH expression

(Hong et al., 2006). In 2008, Hong et al. further discovered that GATA3 also

activates through protein-protein interaction with DBH gene expression in primary

NCSC cultures. GATA3 binds to the transcription factors, Sp1 and AP4, respectively

and the protein complexes bind to the DBH gene promoter to activate transcription

(Hong et al., 2008). Therefore, GATA3 via a novel and distinct protein-protein

interaction directly contributes to noradrenalin phenotype specification.

GATA3 and Tumorigenesis

GATA3 is an important regulator of cellular proliferation and differentiation in

the development. So whether the GATA3 expression in tumor cells has any effect on

tumorigenesis is a question of interest. The effects of GATA3 expression have been

widely investigated in T cell-related tumors and breast cancers.

GATA3 is expressed in human T cell acute lymphoblastic leukemias (T-ALL)

(Minegishi et al., 1997). GATA3 is capable of forming a complex with the 47

transcription factors LIM-only domain protein Lmo2 and the basic-helix-loop-helix

(bHLH) protein Tal1, which are often shown to be aberrant expression pattern in

human T-ALL. Lmo2 and Tal1 act as cofactors for GATA3 to activate the

transcription of retinaldehyde dehydrogenase 2 (RALDH2) gene in T-cell

tumorigenesis (Ono et al., 1998). Antisense GATA3 treatment can inhibit the

expression of TNF-α and Th2 cytokines in tumor cells and also depress tumor growth

in tumor-bearing mice (Yao et al., 2005). Direct evidence for a role of GATA3 in T-

ALL is that enforced expression of GATA3 during the T cell development in CD2-

GATA3 transgenic mice induces CD4+CD8+ double-positive (DP) T cell lymphoma

(Nawijn et al., 2001). Enforced GATA3 expression induces high c-Myc expression

and converts DP thymocytes into a pre-malignant state, whereby subsequent induction

of Notch1 signaling cooperates to establish malignant transformation (van Hamburg

et al., 2008).

The expression of GATA3 is associated with estrogen α receptor in breast

cancers (Hoch et al., 1999). GATA3 is involved in growth control and the maintenance of the differentiated state in mammary epithelial cells, and loss of

GATA3 function may contribute to breast cancer tumorigenesis (Usary et al., 2004).

GATA3 also contributes to the metastasis of breast cancers through its capability to

regulate differentiation. Breast tumors with high GATA3 expression tend to be well

differentiated with low metastatic potential, whereas tumors with low GATA3

expression tend to be poorly differentiated with high metastatic potential (Hoch et al.,

1999; Jacquemier et al., 2005; Jenssen et al., 2002; Mehra et al., 2005). Loss of

GATA3 marks progression from adenoma to early carcinoma and onset of tumor dissemination in breast cancers. Restoration of GATA3 in late carcinomas induces

tumor differentiation and suppresses tumor dissemination. Targeted deletion of

GATA3 in early tumors leads to apoptosis of differentiated cells, which suggests that

the loss of GATA3 is not sufficient for malignant conversion, while an expanding

GATA3-negative tumor cell population accompanies malignant progression. These facts demonstrate that GATA3 regulates tumor differentiation and suppresses tumor dissemination in breast cancers (Kouros-Mehr et al., 2008).

GATA3 has been shown alter its expression patterns in other types of cancers,

such as pancreatic cancers, cervical and esophageal carcinomas. In pancreatic cancers, the mRNA and protein levels of GATA3 are markedly upregulated. The strong specific upregulation of GATA3 impairs nuclear translocation and its cooperative action with the TGF-β pathway, suggesting that GATA3 plays an important role in human pancreatic cancers (Gulbinas et al., 2006). In cervical cancers, a progressive downregulation of GATA3 expression is observed during carcinogenesis. Human papillomavirus-mediated immortalization of cervical cell lines leads to a loss of

GATA3 expression (Steenbergen et al., 2002). In contrast, a clear upregulation of

GATA3 expression is observed in human esophageal cancer cell lines, whereas no expression of GATA3 is observed in normal esophageal mucosal cells and primary carcinoma cells (Shiga et al., 1993).

GATA3 has been shown to express in multiple neuroblastoma cell lines

(George et al., 1994). But no further researches have been investigated.

MANUSCRIPT ONE

Nestin expression defines both glial and neuronal progenitors in

postnatal sympathetic ganglia

Huilin Shi,1 Hongjuan Cui,1 Goleeta Alam,1 William T. Gunning,1,2 Andrea Nestor,2,3

David Giovannucci,2,4 Ming Zhang,5 and Han-Fei Ding1*

1Department of Biochemistry and Cancer Biology, 2Advanced Microscopy and

Imaging Center, 3Department of Surgery, and 4Department of Neurosciences,

University of Toledo Health Science Campus, Toledo, Ohio 43614

5Department of Anatomy and Structural Biology, University of Otago, Dunedin, New

Zealand

Running title: Postnatal sympathetic progenitors

Associate Editor: John L. R. Rubenstein

Key words: noradrenergic neurons; satellite cells; superior cervical ganglia; postnatal sympathetic development

*Correspondence to: Han-Fei Ding, Department of Biochemistry and Cancer Biology,

BHSB 461, University of Toledo Health Science Campus, 3035 Arlington Avenue,

Toledo, Ohio 43614. E-mail: [email protected]

Grant sponsor: the National Cancer Institute; grant number: R01 CA124982.

ABSTRACT

Sympathetic ganglia are primarily composed of noradrenergic neurons and satellite glial cells. Although both cell types originate from neural crest cells, the identities of the progenitor populations at intermediate stages of the differentiation process remain to be established. Here we report the identification in vivo of glial and neuronal progenitor cells in postnatal sympathetic ganglia, using mouse superior cervical ganglia as a model system. There are significant levels of cellular proliferation in mouse superior cervical ganglia during the first 18 days after birth. A majority of the proliferating cells express both nestin and brain lipid-binding protein (BLBP). BrdU fate-tracing experiments demonstrate that these nestin and BLBP double positive cells represent a population of glial progenitors for sympathetic satellite cells. The glial differentiation process is characterized by a marked downregulation of nestin and upregulation of S100, with no significant changes in the levels of BLBP expression.

We also identify a small number of proliferating cells that express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis that defines sympathetic noradrenergic neurons. Together, these results establish nestin as a common marker for sympathetic neuronal and glial progenitor cells and delineate the cellular basis for the generation and maturation of sympathetic satellite cells.

INSTRUCTION

The sympathetic nervous system is composed of sympathetic ganglia and the adrenal

medulla, a specialized sympathetic ganglion containing secretory chromaffin cells.

Sympathetic ganglia of mammals are organized into two paravertebral chains that

span from cervical to sacral regions, with the ganglia being interconnected with pre-

and postganglionic sympathetic nerve fibers. Sympathetic ganglia contain two major

cell types, neurons and glial cells. Most mammalian sympathetic neurons use

noradrenaline as a neurotransmitter and, thus, are called noradrenergic neurons. These

neurons are commonly marked by their expression of tyrosine hydroxylase (TH) that

catalyzes the rate-limiting step in the biosynthesis of catecholamines including

dopamine, noradrenaline, and adrenaline. Sympathetic glial cells include Schwann

cells and satellite cells. Schwann cells provide myelin to insulate axons of the

peripheral nerves whereas satellite cells line the exterior surface of sympathetic neurons. Within a sympathetic ganglion, the majority of glial cells are satellite cells

and Schwann cells are generally associated with intra-ganglionic nerve fibers. A

common marker for the sympathetic glial cells is S100, an acidic calcium-binding

protein (Cocchia and Michetti, 1981).

It is well established that sympathetic neurons and glia are derived from neural crest

cells (Anderson, 1989; LaBonne and Bronner-Fraser, 1998; Le Douarin and Dupin,

1993), a transient, highly migratory population of multipotent stem/progenitor cells.

The neural crest can be divided into four regions along the anterior–posterior axis:

cranial, vagal, trunk, and lumbosacral neural crest. During sympathetic development,

neural crest cells, mainly from the trunk region of the neural crest, migrate ventrally 53

and aggregate adjacent to the dorsal aorta to form the primary sympathetic chain. A

subpopulation of the cells then undergo dorsal migration to form the paravertebral sympathetic ganglia where they differentiate into sympathetic neurons and glial cells

(Francis and Landis, 1999; Kirby and Gilmore, 1976).

The generation of sympathetic neurons (neurogenesis) and glia (gliogenesis), which is

best studied in rat superior cervical ganglia (SCG), occur during different periods of

sympathetic development. While neurogenesis peaks around embryonic day 14.5

(E14.5) and is essentially completed at the time of birth, gliogenesis begins around

E16.5 and continues postnatally (Hall and Landis, 1991; Hall and Landis, 1992;

Hendry, 1977). Consistent with the temporal pattern of in vivo sympathetic

neurogenesis and gliogenesis, in vitro fate tracing experiments revealed that

proliferating cells isolated from the E14.5 rat SCG gave rise predominantly to clones

containing only neurons, whereas those from the E17.5 rat SCG generated mostly

clones that contained only glial cells (Hall and Landis, 1991). These findings have led

to the suggestion that post-migratory neural crest cells commit to a neuronal or glial

fate at a very early stage of the sympathetic development (Hall and Landis, 1991).

However, the identities of sympathetic neuronal and glial progenitors have not been

clearly defined.

In this study, we examined the development of mouse sympathetic ganglia during the

first eight weeks after birth, with the goal of identifying molecular markers that define

distinct sympathetic progenitor populations. A detailed characterization of the cellular

basis for postnatal sympathetic development should facilitate the investigation of genes and signaling pathways that control the developmental process.

MATERIALS AND METHODS

Mice

Male and female C57BL/6J mice 1-day to 8-week old were used in this study. Mice

were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained under

Specific Pathogen Free conditions in the animal facility of University of Toledo

Health Science Campus. All animal experiments were pre-approved by the

Institutional Animal Care and Use Committee.

BrdU injection and SCG collection

The thymidine analog 5-bromo-2’-deoxyuridine (BrdU, Sigma, St. Louis, MO) was

dissolved in saline at 10 mg/ml, and mice were injected intraperitoneally with BrdU at

50 mg/kg (body weight) 2 hours prior to being euthanized by CO2 inhalation on postnatal day 4, or they were injected once daily for 3 days on postnatal day 4, 5 and 6, and sacrificed on day 1, 7, 14, 21, 28, and 56 after the final BrdU injection. C57BL/6J mice without BrdU injection were also euthanized by CO2 inhalation on postnatal day

1, 4, 7, 10, 14, 18, 22, 26, 30, 42 and 56. For each time point, at least 3 mice were

used. Mouse SCG were collected, fixed in 10% neutral buffered formalin and

embedded in paraffin. The paraffin tissue blocks were stored at 4˚C until sectioned.

Immunofluorescence

Sections of 4-µm were cut from the SCG paraffin blocks and mounted on 3- aminopropyltriethoxysilane-treated slides (Sigma). After de-paraffinization and rehydration, the sections were treated in a microwave oven at 95 degree for 20 minutes in 10 mM citrate buffer (pH= 6) for antigen retrieval and washed in PBS.

Sections were blocked with 10% horse serum for 1 hour and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: mouse anti-

Ki67 (1:100, BD Pharmingen, San Jose, CA), rat anti-BrdU (1:200, Abcam,

Cambridge, MA), mouse anti-TH (1:5000, Sigma), rabbit anti-TH (1:1000, Chemicon,

Temecula, CA), rabbit anti-S100 (1:200, Dako, Carpinteria, CA), rabbit anti-BLBP

(1:2000, Chemicon), and chicken anti-nestin (1:1000, Novus Biologicals, Littleton,

CO). For double or triple immunostaining, various combinations of primary antibodies were used. After washing in PBS, the sections were again blocked with

10% goat serum for 1 hour, followed by incubation with secondary antibodies for 2 hours. All secondary antibodies were purchased from Invitrogen (Carlsbad, CA) and used at 1:400 dilutions. The secondary antibodies used were: goat anti-mouse (FITC or Texas-Red), goat anti-rabbit (Alexa fluor 594 or Alexa Fluor 647), goat anti- chicken (Alexa Fluor 488), and goat anti-rat (Alexa Fluor 488). After secondary antibody incubation, the sections were washed in PBS and counterstained with DAPI to stain nuclei. Sections were mounted in Fluorescence Mounting Medium (Dako) and

ProLong Gold antifade reagent (Invitrogen), coverslipped, and examined using a TCS

SP5 multi-photon laser scanning confocal microscope (Leica Microsystems,

Bannockburn, IL) or a Nikon ECLIPSE E800 fluorescence microscope (Nikon

Instruments Inc., Melville, NY). Images were processed using Image J (NIH shareware) and Adobe Photoshop CS (Adobe Systems, Mountain View, CA). Figures were assembled and labeled in Canvas 9 (ACD Systems, Miami, FL). 57

Antibody characterization

The clone B56 anti-Ki67 mouse monoclonal antibody (BD Pharmingen, #550609, Lot

#32659) was raised against human Ki67 protein. It has the same antigen specificity as

the well-characterized clone MIB 1 anti-Ki67 monoclonal antibody (Key et al., 1993).

Both MIB 1 and B56 antibodies recognize a double band of 345 and 395 kD on

Western blot. Flow cytometry analysis reveals that the binding of Phycoerytherin-

conjugated B56 can be blocked by purified MIB 1 antibody. Immunohistochemistry

analysis demonstrates that B56 gives the same staining pattern as MIB 1 on both frozen and paraffin-embedded tissue sections (manufacturer’s Technical Data Sheet).

The specificity of the monoclonal rat anti-BrdU antibody (Abcam, ab6326, Lot

#163833) has been confirmed by several assays. It reacts with free BrdU, BrdU in

single-stranded DNA, or BrdU attached to a carrier protein, but not with thymidine

(manufacturer’s technical information). In our study, no staining was seen when the

antibody was used to stain tissue sections from mice that had not been injected with

BrdU.

The mouse monoclonal anti-TH antibody (clone TH-2, Sigma, T1299) was raised

against rat TH. It recognizes an epitope present in the N-terminal region (amino acids

9-16) of both rodent and human TH. The rabbit polyclonal anti-TH antibody

(Chemicon, AB152, Lot #0603025098) was prepared against denatured TH from rat

pheochromocytoma. Both antibodies detect a single band of ~60 kD on Western blot 58

(manufacturer’s technical information), and only stained sympathetic cells with the

morphology of neurons.

The rabbit polyclonal anti-S100 antibody (Dako, Z0311, Lot #00015317) was raised

against S100 isolated from cow brain. In western blotting of purified recombinant human S100 proteins, the antibody stains S100B strongly, S100A1 weakly, and

S100A6 very weakly. No reaction was observed with S100A2, S100A3 and S100A4

(manufacturer’s technical information). Also according to the information provided

by Dako, Z0311 only labels glial cells in the brain and gives the same staining pattern

as a monoclonal anti-S100 antibody previously reported (Hagen et al., 1986). In our

study, we found that Z0311 only stained cells with the morphology and distribution of

glial cells in adult mouse SCG (Fig. 3).

The rabbit polyclonal anti-BLBP antibody (Chemicon, AB9558, Lot #0603024945)

was raised against recombinant BLBP. It stains a single band of ~15 kD on Western

blot and only glial cells in immunohistochemistry (manufacturer’s technical

information). We also found that the antibody only stained cells with the morphology and distribution of glial cells in mouse SCG (Fig. 2).

The chicken anti-nestin antibody (Novus Biologicals, NB100-1604, Lot #0305) was

raised against mouse nestin. According to the information provided by the company,

the antibody stains neural stem cells of the ventricular zone of the mouse brain by immunofluorescence. In Western blotting, this antibody reacts with recombinant 59

mouse nestin and detects a single band of approximately 250 kD in mouse brain homogenate. No staining was seen in Western blotting of mouse liver homogenate.

We also found that the antibody only stained neural crest cells, but not their neuronal progeny cells (unpublished data), which was identical with previous descriptions

(Stemple and Anderson, 1992).

Quantification

For sections from each mouse at each time point, a total of 1000 cells (DAPI-positive) were counted from at least 4 randomly selected 400x fields, and the percentages of proliferating cells (Ki67- or BrdU-positive) and progenitor- and lineage-marker expressing cells were determined. All data were presented as mean ± standard deviation (SD).

ACKNOWLEDGMENTS

We thank Jane Ding for the assistance in maintaining the mouse colony.

RESULTS

Cell proliferation in postnatal mouse SCG

Progenitor cells are generally defined as populations of dividing cells with the capacity to differentiate (Smith, 2006). To identify the progenitor cell populations in postnatal mouse SCG, we first performed Ki67 immunofluorescence staining of SCG sections (Fig. 1A). Ki67 is a nuclear protein specifically expressed in cells undergoing active proliferation (Sawhney and Hall, 1992). From postnatal day 1 (P1) to P4, approximately 20% of ganglionic cells in mouse SCG expressed Ki67. The number of

Ki67-expressing cells decreased gradually with time, and by the end of the third postnatal week, only 0.4% of ganglionic cells were stained positively for Ki67 (Fig. 1).

Thus, during normal development, sympathetic cell genesis in mice continues at high levels for approximately 2 to 3 weeks after birth. Importantly, the proliferating cells in early postnatal mouse SCG were evenly distributed and no specialized proliferation zones were observed (Fig. 1A). These observations indicate that postnatal development of the sympathetic nervous system is markedly different from that of the central nervous system where continuous neurogenesis persists throughout adulthood in specialized proliferation zones, such as the subventricular zone, the olfactory bulb and the dentate gyrus of the hippocampus (Gross, 2000; Ming and Song, 2005).

Identification of proliferating cells in postnatal mouse SCG

To establish the identities of the Ki67-expressing cells in postnatal mouse SCG, we examined these cells for their expression of lineage markers. For glial markers, we 62

chose S100 and brain lipid-binding protein (BLBP, also called brain fatty acid-

binding protein, BFABP). It has been shown previously that sympathetic satellite cells

express S100 (Cocchia and Michetti, 1981); BLBP is a marker for Schwann cell

precursors and immature Schwann cells of the peripheral nervous system (Jessen and

Mirsky, 2005). We found that all satellite cells in mouse SCG expressed high levels of

BLBP, and the intensity of its expression did not change significantly from P1 to P30

(Fig. 2). This finding indicates that BLBP is an early marker for sympathetic satellite

cells. By contrast, P1 SCG contained no detectable S100-expressing cells, which were

first seen in P4 SCG (Fig. 3, A,B). The number of S100-expressing satellite cells

increased gradually thereafter, and by P22 all satellite cells were stained positively for

S100 (Fig. 3). The expression of S100 was maintained in sympathetic satellite cells

throughout adulthood (data not shown). Thus, S100 appears to be a late marker for

sympathetic satellite cells.

Double immunofluorescence staining and quantification revealed that between P1 and

P18, a period of high levels of postnatal sympathetic cell synthesis, approximately

80% of Ki67-positive cells expressed BLBP (Fig. 2 and 4A), demonstrating that gliogenesis is a predominant event during postnatal sympathetic development. The observation also suggests that early BLBP-expressing satellite cells possess substantial proliferation potential. During the same period, there was a gradual increase in the percentage of Ki67-positive cells that expressed S100, which peaked at the level of ~27% between P14 to P18 (Fig. 3 and 4B). The timing of their appearance suggest that these Ki67 and S100 double positive cells probably represent a subpopulation of immature satellite cells undergoing final rounds of proliferation

before differentiation into mature satellite cells. There were a few Ki67-positive cells

in P22 and P30 SCG that stained negatively for either BLBP or S100 (Fig. 2-4), and

their identity is unknown at the moment.

We also examined Ki67-positive cells for expression of TH, a marker for sympathetic

noradrenergic neurons. We found that approximately 5% of Ki67-positive cells

expressed TH in mouse SCG of P1, P4, and P7 (Fig. 5), indicating that sympathetic

neurogenesis continues at low levels for at least one week after birth. We were not

able to detect consistently Ki67 and TH double positive cells in SCG from older mice

(data not shown). Together, the data from our immunostaining studies suggest that

most proliferating cells in early postnatal mouse SCG are of glial or neuronal lineage.

Differentiation potential of proliferating cells in postnatal mouse SCG

To confirm that the proliferating cells are sympathetic progenitors with the capacity to

differentiate into mature glial cells or neurons, we traced the fates of the cells that were proliferating during the first postnatal week. We injected 4-day-old neonatal

mice with BrdU intraperitoneally once daily for 3 days to label all cycling cells; the

mice were euthanized at various time points after the final BrdU injection. We then

performed triple immunofluorescence staining of SCG sections for BrdU, TH, and

BLBP or S100 to follow the fates of BrdU-labeled cells. Quantification of BrdU-

positive cells at different time points showed a gradual decrease in their numbers,

with approximately 50% of them retaining the BrdU label after 8 weeks (Fig. 6A).

This result suggests that about half of the progenitors continued to proliferate

extensively after BrdU labeling, while the remaining half underwent limited cell divisions and thus retained the BrdU label.

Consistent with our Ki67 studies, we observed that a small number (~5%) of BrdU- positive cells expressed high levels of TH with the morphology of mature neurons in mouse SCG collected within a week after BrdU labeling (Fig. 6B,C), indicating the presence of neuronal progenitors and continuation of neurogenesis in early postnatal mouse sympathetic ganglia.

Staining for BLBP revealed that approximately 80-85% of BrdU-labeled cells expressed BLBP throughout the time points examined (Fig. 6D-J). Using S100 as a marker for glial differentiation, we found that an average of 15% of the BrdU-labeled cells stained positively for S100 one day after the final round of BrdU injection (Fig.

6D,K). Thereafter, there was a gradual increase in the number of BrdU and S100 double positive cells. The number peaked by the end of 3 weeks after BrdU labeling, with ~80% of BrdU-labeled cells expressing S100 (Fig. 6D,K-P). These findings indicate that most of the ganglionic cells that were proliferating during the first postnatal week are BLBP-expressing satellite cell progenitors, which eventually differentiate into S100-expressing satellite cells. The data from our fate-tracing studies also provide further evidence that the generation of satellite cells is a predominant process of postnatal sympathetic development.

Nestin expression marks both glial and neuronal progenitor cells in postnatal

mouse SCG

The postnatal sympathetic glial and neuronal progenitors express the lineage markers

BLBP and TH, respectively. However, neither marker is specifically associated with

sympathetic progenitor cells, as BLBP expression is maintained in differentiated

satellite cells and TH in mature neurons. To understand the molecular mechanisms

that control the progenitor cell differentiation during postnatal sympathetic

development, it is essential to identify the molecular markers whose expression marks

distinct stages of the developmental process. We focused our studies on nestin, a

filament protein initially identified as a maker for neural stem cells in the central

nervous system (Lendahl et al., 1990). Importantly, it has been shown previously that

nestin is expressed in neural crest cells from which all sympathetic neurons and glia

originate (Stemple and Anderson, 1992).

Immunofluorescence staining revealed that a large number of cells in P1 and P4 SCG

expressed significant levels of nestin, which often formed a thin layer of filamentous

structure around nuclei (Fig. 7A,B). Nestin expression was gradually downregulated

thereafter and was largely undetectable around P18 (Fig. 7C-F). This temporal pattern of nestin expression closely matches that of Ki67 (Fig. 1). Indeed, double immunofluorescence staining for Ki67 and nestin revealed that approximately 92-

93% of Ki67-positive cells in P1 and P4 SCG also expressed nestin (Fig. 7G,H,I). The percentage of Ki67 and nestin double positive cells decreased with time, and by P18 only about 13% of Ki67-positive cells showed detectable levels of nestin expression

(Fig. 7I). Given that most of Ki67-positive cells in P1 SCG differentiated into glia or 66

neurons by P18 (Fig. 2-5), the Ki67+nestin- cells in P7-P18 SCG probably represent a

population of proliferating cells undergoing differentiation or immature glial cells and

neurons. Taken together, our data suggest that nestin is a specific marker for

sympathetic progenitor cells.

We next determined whether nestin expression is specific for glial or neuronal

progenitor cells. We injected mice at P4 with BrdU and euthanized them 2 hours later

to minimize the chance of BrdU-labeled cells to undergo extensive differentiation.

Triple immunofluorescence staining of SCG sections was performed with antibodies

against BrdU, nestin, and BLBP or TH, and the sections were examined with a

confocal microscope. Consistent with the results of our Ki67 and nestin double staining experiments, approximately 95% of the BrdU-labeled cells expressed nestin

(data not shown). Importantly, almost all of BrdU- and nestin-positive cells that were examined expressed either BLBP (Fig. 7J,K) or TH (Fig. 7L,M). These findings demonstrate that nestin is a marker for both glial and neuronal progenitor cells.

DISCUSSION

In this paper, we report three major findings from our in vivo examination of the postnatal development of mouse sympathetic ganglia, using mouse SCG as a model system. First, we show that nestin is a molecular marker for both sympathetic glial

and neuronal progenitors. Second, we identify a population of nestin and BLBP

double positive cells as the direct precursors of sympathetic satellite cells. Third, our

study reveals a temporal expression pattern of glial lineage markers that defines the

cellular intermediates in the process of satellite cell differentiation (Fig. 8). These

findings define the cellular basis of postnatal sympathetic development, which should

facilitate the investigation of the genes and signaling pathways that regulate the

developmental process.

Nestin as a marker for both glial and neuronal progenitors in postnatal

sympathetic ganglia

Nestin is an intermediate filament originally identified in neural stem cells (Lendahl et

al., 1990). In the central nervous system, nestin expression marks the proliferating

state of neurogenesis and is rapidly downregulated during differentiation (Dahlstrand

et al., 1995). Neural crest cells isolated from rat embryonic trunk neural tubes have

also been shown to express nestin (Stemple and Anderson, 1992), which give rise to

both neurons and glia of the sympathetic nervous system (Le Douarin and Kalcheim,

1999). Our study reveals that during postnatal sympathetic development, nestin

expression levels are at the highest at P1 and gradually decline thereafter, becoming

largely undetectable by P18. This temporal expression of nestin correlates closely

with the proliferating phase of postnatal sympathetic development. In addition, triple

immunofluorescence staining of multiple lineage markers and BrdU-based fate-

tracing experiments show that nestin is expressed in both glial-restricted and neuron-

restricted progenitor cells. These observations, coupled to the previously reported

nestin expression in neural crest cells (Stemple and Anderson, 1992), identify nestin

as a molecular link between neural crest cells and sympathetic progenitors. Also

importantly, our findings provide nestin as a molecular marker for identifying genes

and signaling pathways that promote the differentiation and maturation of sympathetic

neurons and satellite cells.

The identities of neuronal and glial progenitors in postnatal sympathetic ganglia

Noradrenergic neurons and satellite glial cells are the two main cell types that

constitute sympathetic ganglia. The progenitors for sympathetic neurons have been extensively studied. It is generally accepted that both sympathoadrenal progenitors

from the trunk neural crest and sympathoenteric progenitors from the anterior vagal

neural crest contribute to the generation of sympathetic neurons (Anderson, 1993;

Birren and Anderson, 1990; Carnahan and Patterson, 1991; Durbec et al., 1996;

Francis and Landis, 1999). These progenitors are characterized by their nonneuronal morphology and expression of neurofilaments (NF68 and NF160) and c-Ret

(Guillemot et al., 1993; Sommer et al., 1995), a protein tyrosine kinase co-receptor for glial cell-derived neurotrophic factor (Plaza-Menacho et al., 2006). It is not known

whether these early neuronal progenitors are present in postnatal sympathetic ganglia.

Our study reveals that in postnatal mouse SCG, approximately 5% of proliferating

cells express nestin and TH. These cells are morphologically indistinguishable from 69

the rest of post-mitotic sympathetic neurons, suggesting that they may represent a population of progenitor cells late in the neurogenic process.

In contrast, very little is known about the identity of the glial progenitors that give rise to sympathetic satellite cells. Using Ki67 to mark cells in an active proliferation state, a property of progenitor cells, we found that the vast majority of Ki67-positive cells in postnatal mouse SCG express BLBP, a nervous system-specific member of the lipid- binding protein family that was first identified in radial glial cells and immature astrocytes of the central nervous system (Feng et al., 1994; Kurtz et al., 1994). Our

BrdU fate-tracing experiments further show that the BLBP-positive proliferating cells eventually differentiate into S100-expressing satellite cells, demonstrating that they function as progenitors of sympathetic satellite cells. However, BLBP is not an exclusive marker of the glial progenitors, as mature satellite cells maintain high levels of BLBP expression. In contrast to BLBP, nestin is expressed at high levels in proliferating glial progenitors but is markedly downregulated in differentiated satellite cells. Also, very few S100-positive satellite cells express detectable levels of nestin

(data not shown). Thus, nestin and BLBP expression defines a population of satellite progenitors in mouse postnatal sympathetic ganglia.

Postnatal sympathetic development

The embryonic development of mammalian sympathetic ganglia has been extensively studied, which in rat SCG is characterized by the generation of neurons which peaks around E14.5, followed by a wave of gliogenesis that begins around E16.5 (Hall and

Landis, 1991; Hall and Landis, 1992; Hendry, 1977). For postnatal sympathetic

development, the predominant event is the proliferation of nonneuronal cells, which,

in rat SCG, lasts for at least two weeks after birth (Hendry, 1977). We confirm this observation in postnatal mouse SCG, which shows significant levels of proliferation

up to 18 days after birth. We further demonstrate that, based on both morphology and expression of lineage markers, the vast majority of the proliferating cells are glial progenitors that give rise to satellite cells. Thus, the continuing generation and further

differentiation of satellite cells characterize the postnatal development of mouse

sympathetic ganglia.

During the postnatal gliogenic process in mouse SCG, both glial progenitors and mature satellite cells maintain high levels of BLBP expression, whereas nestin expression is downregulated and S100 expression is upregulated (Fig. 8A). Based upon lineage marker expression profiles and proliferation states, we suggest a model for the glial cell populations that may represent distinct stages of sympathetic gliogenesis (Fig. 8B). The glial progenitors are defined by the expression of both

nestin and BLBP, and they possess the capacity to undergo multiple rounds of

proliferation. The mature satellite cells are defined by the expression of both BLBP and S100, and they are post-mitotic cells. There is also a population of BLBP-positive glial cells with no or low levels of nestin and S100 expression, which may represent immature satellite cells with limited proliferation potential. The identification of the distinct glial populations and the developmental stages that they represent should help

uncover the genes and signaling pathways that control the generation of satellite cells

in the sympathetic nervous system.

LITERATURE CITED

Anderson, D. J. (1989). The neural crest cell lineage problem: neuropoiesis? Neuron 3,

1-12.

Anderson, D. J. (1993). Cell fate determination in the peripheral nervous system: the

sympathoadrenal progenitor. J Neurobiol 24, 185-198.

Birren, S. J., and Anderson, D. J. (1990). A v-myc-immortalized sympathoadrenal

progenitor cell line in which neuronal differentiation is initiated by FGF but not NGF.

Neuron 4, 189-201.

Carnahan, J. F., and Patterson, P. H. (1991). Isolation of the progenitor cells of the

sympathoadrenal lineage from embryonic sympathetic ganglia with the SA

monoclonal antibodies. J Neurosci 11, 3520-3530.

Cocchia, D., and Michetti, F. (1981). S-100 antigen in satellite cells of the adrenal

medulla and the superior cervical ganglion of the rat. An immunochemical and

immunocytochemical study. Cell Tissue Res 215, 103-112.

Dahlstrand, J., Lardelli, M., and Lendahl, U. (1995). Nestin mRNA expression

correlates with the central nervous system progenitor cell state in many, but not all,

regions of developing central nervous system. Brain Res Dev Brain Res 84, 109-129.

Durbec, P. L., Larsson-Blomberg, L. B., Schuchardt, A., Costantini, F., and Pachnis,

V. (1996). Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. Development 122, 349-358.

Feng, L., Hatten, M. E., and Heintz, N. (1994). Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS. Neuron 12, 895-908.

Francis, N. J., and Landis, S. C. (1999). Cellular and molecular determinants of sympathetic neuron development. Annu Rev Neurosci 22, 541-566.

Gross, C. G. (2000). Neurogenesis in the adult brain: death of a dogma. Nat Rev

Neurosci 1, 67-73.

Guillemot, F., Lo, L. C., Johnson, J. E., Auerbach, A., Anderson, D. J., and Joyner, A.

L. (1993). Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75, 463-476.

Hagen, E. C., Vennegoor, C., Schlingemann, R. O., van der Velde, E. A., and Ruiter,

D. J. (1986). Correlation of histopathological characteristics with staining patterns in human melanoma assessed by (monoclonal) antibodies reactive on paraffin sections.

Histopathology 10, 689-700.

Hall, A. K., and Landis, S. C. (1991). Early commitment of precursor cells from the rat superior cervical ganglion to neuronal or nonneuronal fates. Neuron 6, 741-752.

Hall, A. K., and Landis, S. C. (1992). Division and migration of satellite glia in the embryonic rat superior cervical ganglion. J Neurocytol 21, 635-647.

Hendry, I. A. (1977). Cell division in the developing sympathetic nervous system. J

Neurocytol 6, 299-309.

Jessen, K. R., and Mirsky, R. (2005). The origin and development of glial cells in peripheral nerves. Nat Rev Neurosci 6, 671-682.

Key, G., Becker, M. H., Baron, B., Duchrow, M., Schluter, C., Flad, H. D., and

Gerdes, J. (1993). New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3)

generated against bacterially expressed parts of the Ki-67 cDNA containing three 62

base pair repetitive elements encoding for the Ki-67 epitope. Lab Invest 68, 629-636.

Kirby, M. L., and Gilmore, S. A. (1976). A correlative histofluorescence and light

microscopic study of the formation of the sympathetic trunks in chick embryos. Anat

Rec 186, 437-449.

Kurtz, A., Zimmer, A., Schnutgen, F., Bruning, G., Spener, F., and Muller, T. (1994).

The expression pattern of a novel gene encoding brain-fatty acid binding protein

correlates with neuronal and glial cell development. Development 120, 2637-2649.

LaBonne, C., and Bronner-Fraser, M. (1998). Induction and patterning of the neural

crest, a stem cell-like precursor population. J Neurobiol 36, 175-189.

Le Douarin, N. M., and Dupin, E. (1993). Cell lineage analysis in neural crest ontogeny. J Neurobiol 24, 146-161.

Le Douarin, N. M., and Kalcheim, C. (1999). The neural crest, Vol 36, 2nd edn

(Cambridge, Cambridge University Press).

Lendahl, U., Zimmerman, L. B., and McKay, R. D. (1990). CNS stem cells express a new class of intermediate filament protein. Cell 60, 585-595.

Ming, G. L., and Song, H. (2005). Adult neurogenesis in the mammalian central

nervous system. Annu Rev Neurosci 28, 223-250.

Plaza-Menacho, I., Burzynski, G. M., de Groot, J. W., Eggen, B. J., and Hofstra, R. M.

(2006). Current concepts in RET-related genetics, signaling and therapeutics. Trends

Genet 22, 627-636.

Sawhney, N., and Hall, P. A. (1992). Ki67--structure, function, and new antibodies. J

Pathol 168, 161-162.

Smith, A. (2006). A glossary for stem-cell biology. Nature 441, 1060.

Sommer, L., Shah, N., Rao, M., and Anderson, D. J. (1995). The cellular function of

MASH1 in autonomic neurogenesis. Neuron 15, 1245-1258.

Stemple, D. L., and Anderson, D. J. (1992). Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell 71, 973-985.

FIGURE LEGENDS

Fig. 1. Identification of proliferating cells in postnatal mouse SCG. A-J:

Immunofluorescence staining for Ki67-positive cells (red) in SCG from P1 to P56.

Sections were counterstained with DAPI (blue) to visualize nuclei. There were significant levels of proliferation in SCG during the first 18 days after birth. Scale bar

= 100 µm. K: Quantification of Ki67-postive cells in SCG from P1 to P30. At least

1000 cells were counted from each ganglion and 3-4 ganglia were examined for each time point. Data are presented as mean ± SD.

Fig. 2. BLBP immunofluorescence staining for lineage identification of proliferating cells in postnatal mouse SCG. Sections of SCG from P1 to P30 were stained with antibodies against Ki67 (green) and BLBP (red). All satellite cells stained positively for BLBP. The majority of Ki67-positive cells expressed BLBP (yellow), indicating that the postnatal development of sympathetic ganglia is predominantly a gliogenic process that was essentially completed by P22. Scale bar = 50 µM.

Fig. 3. S100 immunofluorescence staining for lineage identification of proliferating cells in postnatal mouse SCG. Sections of SCG from P1 to P30 were stained with antibodies against Ki67 (green) and S100 (red). Postnatal sympathetic development in

SCG is characterized by a gradual increase in the number of S100-expressing satellite cells, which peaked by P22. In SCG from P7 to P18, a subpopulation of Ki67-positive cells expressed S100 (yellow). Scale bar = 50 µM. 76

Fig. 4. Quantification of Ki67-positive cells that express the glial lineage marker

BLBP or S100. For each time point, 3-4 ganglia were examined, and Ki67 single

positive and Ki67-BLBP (A) or Ki67-S100 (B) double positive cells were counted

from at least 4 randomly selected fields (400X). Data are presented as mean ± SD.

Fig. 5. TH immunofluorescence staining for lineage identification of proliferating

cells in postnatal mouse SCG. Sections of SCG from P1 to P7 were stained with antibodies against Ki67 (green) and TH (red), and counterstained with DAPI (blue) to visualize nuclei. Arrows indicate Ki67 and TH double positive cells, which represented approximately 5% of Ki67-positive cells. The Ki67-positive neurons are

morphologically indistinguishable from the rest of post-mitotic neurons. Scale bar =

25 µM.

Fig. 6. BrdU fate-tracing study of proliferating cells in postnatal mouse SCG. Mice at

P4 were injected with BrdU once daily for 3 days and sacrificed on day (D) 1, 7, 14,

21, 28, 56 after the final round of BrdU injection. Mouse SCG sections were stained

with antibodies against BrdU (green), TH (blue), and BLBP or S100 (red). A:

Quantification of BrdU label-retaining cells in SCG from D1 to D56. For each time

point, 3-4 ganglia were examined. BrdU-positive cells were counted from at least 4

randomly selected fields (400X) and the average number of BrdU-positive cells per

field from D1 SCG was defined as 100%. Data are presented as mean ± SD. B,C:

Confocal immunofluorescence images showing postnatal mouse SCG at D1 (B) and

D7 (C) contained BrdU and TH double positive neurons (arrows), which account for

approximately 5% of total BrdU-positive cells. Scale bar = 25 µm. D: Quantification

of BrdU-positive cells that express the glial lineage (Lin+) marker BLBP or S100 at

the indicated time points. For each time point, 3-4 ganglia were examined. BrdU single positive and BrdU-BLBP or BrdU-S100 double positive cells were counted

from at least 4 randomly selected fields (400X). Data are presented as mean ± SD. E-

P: Confocal immunofluorescence images of SCG sections from mice at different time

points (D1-D56) following the final round of BrdU injection. The sections were

stained with antibodies to BrdU, TH, and BLBP (E-J) or S100 (K-P), showing that

BrdU-labeled, BLBP single-positive cells in D1 SCG differentiated into satellite cells

expressing both BLBP and S100 in a time-dependent manner. Scale bars = 50 µm.

Fig. 7. Nestin is a common marker for glial and neuronal progenitors in postnatal mouse SCG. A-F: Time-course study of nestin expression in mouse SCG from P1 to

P18. Sections were stained with an antibody against nestin (green), counterstained with DAPI (blue), and examined with a confocal microscope. Nestin expression was essentially undetectable by P18. Scale bar = 50 µm. G,H: Confocal

immunofluorescence images of SCG sections from mice at P1 (G) and P4 (H). The sections were stained with antibodies against nestin (green) and Ki67 (red), showing nestin expression in Ki67-positive proliferating cells. Scale bar = 25 µm. I:

Quantification of Ki67 and nestin double positive cells in mouse SCG at the indicated time points (P1-P18). For each time point, 3 ganglia were examined. Ki67 single- positive and Ki67-nestin double positive cells were counted from 3 randomly selected fields (400X). Data are presented as mean ± SD. J-M: Confocal immunofluorescence

images of SCG sections from mice at P4. The mice were injected with BrdU and sacrificed after 2 hours. J,K: Sections were stained with antibodies against BrdU

(blue), nestin (green) and BLBP (red), showing nestin expression in BrdU and BLBP double positive glial progenitors (arrows). Scale bar = 25 µm. L,M: Sections were stained with antibodies against BrdU (blue), nestin (green), and TH (red), showing nestin expression in BrdU and TH double positive neuronal progenitors (arrows).

Scale bar = 25 µm.

Fig. 8. A model for the generation and differentiation of satellite cells in postnatal sympathetic ganglia. A: A diagram for the temporal expression pattern of molecular markers (nestin, BLBP and S100) during postnatal sympathetic gliogenesis. B: A model for the putative glial cell populations that represent distinct stages of satellite cell differentiation (see text for detail).

MANUSCRIPT TWO

GATA3 regulation of human neuroblastoma stem cell activities

Huilin Shi1, Hongjuan Cui1, Goleeta Alam1, Jane Ding2, Jean H. Overmeyer1, William A. Maltese1, Han-Fei Ding2*

1Department of Biochemistry and Cancer Biology,

University of Toledo, Health Science Campus,

Toledo, Ohio 43614, USA.

2Department of Pathology and Cancer Center,

Medical College of Georgia,

Augusta, GA 30912, USA.

*Corresponding author: Han-Fei Ding [email protected]

Subject categories: Cancer initiation

Abstract

Neuroblastoma is a common childhood malignant tumor of the sympathetic nervous system. BE(2)-C cells, a human neuroblastoma cell line enriched with tumorigenic stem cells, can be induced to undergo either neuronal or glial differentiation. We use

BE(2)-C cells as a system to identify the genes that regulate neuroblastoma stem cell activities, such as self-renewal and differentiation. One of these genes is GATA3, a zinc-finger transcription factor with an essential role in the sympathetic development.

Downregulation of GATA3 by siRNA promotes BE(2)-C cell proliferation and overexpression of GATA3 decreases the proliferation of BE(2)-C cells. GATA3 regulates cell proliferation through distinct pathways involving Cyclin D1 and E2F1, as evidenced by an increased expression of Cyclin D1 and a decreased expression of

E2F1 in GATA3 knockdown cells. In addition, GATA3 knockdown induces BE(2)-C cells to undergo glial differentiation, as indicated by an increase in the expression of

GFAP, a glial cell marker, and a decrease in the expression of neuronal marker

SNAP25. GATA3 overexpression promotes neuronal differentiation of BE(2)-C cells, as indicated by a reduction of GFAP expression and an upregulation of SNAP25 expression. GATA3 knockdown also downregulates Phox2b and GATA3 overexpression upregulates Mash1, two transcription factors that are expressed in neuronal progenitor cells and are essential for the sympathetic development. Together, these findings suggest that GATA3, Phox2b and Mash1 may function in a regulatory network in the control of neuroblastoma cells in a stem/progenitor cell state.

Keywords: Neuroblastoma/Cancer stem cell/GATA3/BE(2)-C/Cyclin D1/E2F1

Introduction

Neuroblastoma is a common childhood solid cancer and originated from neural crest

stem cells (NCSCs) or sympathetic precursor cells (Brodeur, 2003), occurring in

sympathetic ganglia and adrenal medulla. 90% of children with this disease are

diagnosed before the age of 6 years (Schwab et al., 2003). Neuroblastoma can either

regress spontaneously, usually in infants, or mature into a benign ganglioneuroma by

undergoing apoptosis and/or differentiation. However in children over 1 year old, the

tumors are mostly aggressive and fatal and their overall prognosis has been poor

(Brodeur, 2003; Nakagawara, 1998). The enigmatic clinical behavior of

neuroblastoma is based on its heterogeneous property. Histopathologically,

neuroblastoma is characterized by heterogeneous composition, from tumors

consisting of dominant undifferentiated neuroblasts to those comprised of largely

fully differentiated neurons with a dense stroma of Schwann cells (Castleberry, 1997).

The cell lines established from human neuroblastomas also exhibit heterogeneity.

Three distinct types of cell lines have been identified based on their morphologies, growth patterns and biochemical properties. They are N for neuroblastic, S for nonneuronal, substrate-adherent and I for intermediate cell types (Ross et al., 2003). N-

type cells have immature neuroblastic features with small and rounded bodies, a high

nuclear to cytoplasmic ratio and short neurites. They attach better to other cells than

to substrate and form cell aggregates in culture. They express neurofilaments, specific

noradrenergic enzymes and receptors (Ross et al., 2002). S-type cells show 90

characteristics opposite to those of N-type cells. They are large and flattened cells

with abundant cytoplasm. They may have long filopodia but no neurites. These cells

adhere tightly to substrate, grow as a monolayer in culture and present contact

inhibition of growth. They express glial cell markers like vimentin or glial fibrillary

acidic protein (GFAP) (Acosta et al., 2009). I-type cells exhibit an intermediate status

between N-type and S-type cells. They attach equally well to both the substrate and to

other cells. They may or may not have neurites and form multilayers with focal

aggregates in cultures. They have nuclei similar to N-type cells and more cytoplasm

like S-type cells. I-type cells express both N- and S- type cell markers and stem cell

markers, such as CD133 and c-kit (Walton et al., 2004).

It has been proposed that I-type cells may represent a population of neuroblastoma

stem cells based on their differentiation and clonogenic ability. I-type cells can

differentiate into both N-type and S-type cells under different conditions (Acosta et al.,

2009). N-type cells can be induced to neuronal or neuroendocrine cells (Ross et al.,

2002) whereas S-type cells can further differentiate into Schwann/glial cells,

melanocytes and smooth muscle cells (Slack et al., 1992; Sugimoto et al., 2000). I-

type cells have the greatest clonogenic activity in soft agar and tumorigenic potential

in immunodeficient mice. S-type cells are non-tumorigenic and N-type cells exhibit tumorigenic ability depending on particular cell lines (Spengler et al., 1997).

Neuroblastoma is one of few cancers originating during the embryonic development, which implies that this tumor may be initiated from transformed developing NCSCs or malignant sympathetic precursor cells that obtain stem cell properties through mutations. This neuroblastoma stem cell model can adequately interpret the

heterogeneous characteristic of human neuroblastoma tumors, based on the self-

renewal and differentiation ability of malignant NCSCs or sympathetic precursor cells.

I-type neuroblastoma cell lines have been identified as cancer stem cells based on

their special phenotypic characterization, differentiation potential and malignant

potential (Ross and Spengler, 2007).

Since neuroblastoma cancer stem cells play an essential role in the tumor initiation, it

is necessary to investigate the regulatory mechanisms underlying self-renewal and

differentiation capacities of neuroblastoma stem cells, which provide the foundation

for appropriate future therapies in neuroblastomas. In this study, we investigated the

GATA3 regulation of BE(2)-C cell – an I-type neuroblastoma cell line – activities,

including tumorigenicity and differentiation. In vertebrates, the GATA family contains 6 six members (named GATA1 to 6) which belong to zinc finger transcription factors and bind to the common consensus DNA sequence

(A/T)GATA(A/G) (Patient and McGhee, 2002). GATA3 plays an essential role in the development of many tissues in vertebrates, including Th2 cell differentiation, the development of the kidney, the inner ear, embryonic and pubertal mammary glands,

adipocytes and neurons, et al. (Asselin-Labat et al., 2007; Grote et al., 2008; Kouros-

Mehr et al., 2008; Lillevali et al., 2006; Smith et al., 2002; Tong et al., 2005; van

Doorninck et al., 1999; Yamashita et al., 2005). The haploinsufficiency of the

GATA3 gene in chromosome 10p causes HDR (hypoparathyroidism, deafness and

renal dysplasia) syndrome (Van Esch et al., 2000). In the sympathetic nervous system,

GATA3-/- mouse embryos die by embryonic day 11 (E11) due to noradrenaline

deficiency. Loss of GATA3 leads to dramatic downregulation of the TH and DBH

expression (Lim et al., 2000). GATA3 controls sympathoadrenal differentiation by

mutual modulation with the other sympathetic regulators Mash1, Phox2b and dHand

(Moriguchi et al., 2006). GATA3 also contributes to tumorigenesis in multiple tumors,

such as T cell acute lymphoblastic leukemias (T-ALL) and breast cancers (Minegishi

et al., 1997; Usary et al., 2004). Enforced expression of GATA3 during the T cell

development in CD2-GATA3 transgenic mice induces CD4+CD8+ double-positive

(DP) T cell lymphoma (Nawijn et al., 2001). Loss of GATA3 in breast cancers induces poorly differentiated tumors cells and promotes dissemination (Kouros-Mehr

et al., 2008). Therefore, it is a question of interest to investigate the role of GATA3 in the regulation of neuroblastoma stem cell activities to uncover the mechanisms underlying tumorigenesis of neuroblastoma.

Results

Loss of GATA3 increases the clonogenic capacity of BE(2)-C cells

Cancer cells can grow in a semi-solid medium, such as soft agar or methylcellulose

whereas non-cancer cells grow only when attached to a surface. The semi-solid

medium can prevent the migration of cells and lead to the formation of spatially

distinct colonies (Thomson and Meyskens, 1982). The anchorage-independent growth

is an important characteristic of cancer cells and is correlated closely with the

tumorigenic capacity of cancer cells. BE(2)-C-pSIH1-H1-puro-GFPsi (abbr BE(2)-C-

GFPsi) and BE(2)-C-pSIH1-H1-puro-GATA3si (abbr BE(2)-C-GATA3si) cells were

plated at 1000 cells per well (6-well culture plates). Viable colonies were stained with

MTT 11 days after plating. By dividing the number of colonies by the number of cells

plated, it is estimated that the colonies formed by BE(2)-C-GATA3si cells are higher

than that of BE(2)-C-GFPsi by 44.9% (Figure 1AB). Therefore, loss of GATA3

expression significantly increases the cloning efficiency in soft agar and increases the

clonogenic capacity of BE(2)-C cells. The soft agar colony formation assay performed in GATA3 overexpression cells further confirms this conclusion. The colonies formed

by BE(2)-C-pBabe-puro-GATA3 cells are 31.85% fewer than those formed by BE(2)-

C-pBabe-puro cells (Figure 1CD), which suggests that the function of GATA3 is to

suppress the clonogenic capacity of BE(2)-C cells.

Clonogenic assays were performed by plating 1000 cells in a 100mm plate and

staining with 0.4% crystal violet after 14 days. Based on the percentage of the number 94

of colonies formed in that of plated cells, BE(2)-C-GATA3si cells give rise to more

colonies than BE(2)-C-GFPsi cells by 57.55%, whereas BE(2)-C-pBabe-puro-

GATA3 cells produce fewer colonies than those of BE(2)-C-pBabe-puro cells by

26.65% (Figure 2). Clonogenic assay reflects long term cell survival of the specific

cell line, but can’t exhibit the clonogenic capacity of the cell line because this

technique is feasible to any attached cells including non-cancer cells and cancer cells.

The crystal violet staining is chemical staining and is not specific for viable cells.

Even though, these results still indicate that loss of GATA3 increases the proliferative

potential of BE(2)-C cells.

In the MTT assay, the number of viable cells is reflected by the OD value. The viable

cell numbers of the GATA3 knockdown or overexpression cell line were measured in continuous 7 days in cultures at initially 500 or 750 cells. There is an increase in the

number of viable cells in the cultures of BE(2)-C-GATA3si cells and a decrease in the

BE(2)-C-pBabe-puro-GATA3 cell cultures compared with their respective controls

(Figure 3). Though the MTT assay can reflect the dynamic process for proliferative

potentials of cells, it still has technical limitations which are caused by the ratio of

MTT amount to cell numbers, culture time, the frequency for medium change, et al.

Therefore, the cell growth assay was performed to confirm the MTT results. In this

assay, the exact cell numbers in cultures at indicated time points were examined,

which compensates the limitations of the MTT assay and reflects the dynamic process

of cell proliferation. As shown in Figure 4, the growth rate of GATA3 knockdown

cells is markedly increased compared with control cells, whereas a decreased rate of

cell proliferation is detected in GATA3 overexpression cells. To summarize, these

four assays are highly consistent and demonstrate that loss of GATA3 increases the clonogenic capacity and proliferative potential of BE(2)-C cells.

Cyclin D1 and E2F1 are the downstream genes of GATA3 responsible for the increased clonogenicity of BE(2)-C cells

Since the loss of GATA3 increases the proliferative potential of BE(2)-C cells, it is interesting to investigate the mechanisms underlying the increased clonogenic capacity. The expression of candidate downstream genes was investigated in the

GATA3 knockdown cell line and its control.

The first group of genes - p16, E2F1 and the retinoblastoma protein (pRb) - is the regulators closely involved in controlling the late G1 phase checkpoint in the cell cycle. Cyclin D/CDK4 or Cyclin D/CDK6 complexes can phosphorylate pRb and the phosphorylated pRb releases E2F transcription factors which regulate gene transcription required for DNA synthesis and promote cells to enter into S phase irreversibly (Diehl, 2002; Weinberg, 1995). The p16 functions as a cyclin-dependent kinase (CDK) inhibitor and specifically binds to CDK4 and CDK6 thus leading to G1 arrest by inhibiting their kinase activity (Aprelikova et al., 1995). In our research, p16 is expressed equally in the GATA3 knockdown and control cells (Figure 5B), which suggests that GATA3 is not acting through p16 to regulate cell proliferation and p16 is not responsible for the expression alteration of pRb and E2F1. The pRb expression is dramatically upregulated in GATA3 knockdown cells compared with the control

(Figure 5B). Because pRb has two forms, phosphorylated and unphosphorylated, it’s

difficult to distinguish the difference in the expression alteration of these two forms

using general pRb antibodies. Therefore, the upregulation of pRb reflects the change

of total pRb expression and implies that it may be a downstream gene of GATA3 to

promote cell cycle progression. The expression pattern of E2F1 in our research is not

consistent with the reported function of this gene in promoting cell proliferation

through the Cyclin D-pRb-E2F pathway. The E2F1 expression is significantly

downregulated in GATA3 knockdown cells compared with the control (Figure 5B).

However, the E2F family also includes other proteins, such as E2F2 to E2F8; E2F3a

is also a downstream target of pRb promoting the cell cycle (Chong et al., 2009). The

overexpression of E2F1 has been proven to increase apoptosis (Wu and Levine, 1994).

Therefore, our results are consistent with the decreased E2F1 expression causing an

increased clonogenicity of BE(2)-C cells through apoptosis suppression.

The second group of genes - p27, p21 and p53 - belongs to tumor suppressor genes

while the p21 and p27 are CDK inhibitors, which play important roles as negative

regulators during progression of the cell cycle. The p27 can bind to the Cyclin

D/CDK4 complex and prevent CDK4 from phosphorylating its substrate - pRb. The

increased level of p27 typically causes cells to arrest in the G1 phase of the cell cycle.

The p27 can also bind to the Cyclin A/CDK2 and Cyclin E/CDK2 complexes

(Toyoshima and Hunter, 1994). The p21 expression is induced by p53 during a DNA

damage-induced G1-phase checkpoint response and inhibits both CDK4 and CDK2 activities (He et al., 2005). The loss of the CDK inhibitors occurs in multiple tumors.

The p27 mutation has been proven to exist in the breast, pituitary and colon cancers

(Galizia et al., 2004; Spirin et al., 1996; Takeuchi et al., 1998), et al. The decreased

expression of p21, p27 and p53 is expected to occur in poor prognosis cancers

(Klopfleisch and Gruber, 2009; Koljonen et al., 2006). In our research, the expression

levels of p21, p27 and p53 in GATA3 knockdown cells are not significantly different from control cells (Figure 5C). This implies that these CDK inhibitors are not downstream targets of GATA3 responsible for the increased proliferation while they

are not involved in the regulation of the pRb or E2F1 level.

Cyclins are a family of proteins which control the progression of cells through the cell

cycle by regulation of CDKs (Galderisi et al., 2003). Cyclins themselves have no enzymatic activity. There are two main groups of cyclins: G1/S cyclins are essential

for the control of G1/S transition in the cell cycle; G2/M cyclins are required for the

G2/M transition. G1/S cyclins include Cyclin A/CDK2, Cyclin D/CDK4, Cyclin

D/CDK6 and Cyclin E/CDK2 while G2/M cyclins contain Cyclin B/CDK1 (Galderisi

et al., 2003). The overexpression of cyclins promotes cell cycle progression and

occurs in diverse cancers (Collecchi et al., 2000; Knudsen et al., 2006; Lotayef et al.,

2000; Schildkraut et al., 2008; Wang et al., 1997). In our research, GATA3 deficiency

induces the upregulation of Cyclin D1 (Figure 5D), which suggests that Cyclin D1 is

responsible for the increased proliferative potential and is the downstream target of

GATA3. GATA3 suppresses the cell proliferation through inhibiting the Cyclin D1

expression. In other three cyclins, Cyclin B1 and Cyclin E are equally expressed

between the GATA3 knockdown cell line and its control whereas the Cyclin A

expression is significantly decreased under GATA3 deficiency, which is inconsistent

with the context (Figure 5D).

GATA3 expression is closely related to the differentiation status of BE(2)-C cells

The essential role of GATA3 in the sympathetic nervous system is to induce the differentiation of sympathetic neurons by promoting TH synthesis. Loss of GATA3 will result in mouse lethality around E11 due to noradrenergic deficiency (Moriguchi et al., 2006). Since BE(2)-C cells are potential neuroblastoma stem cells and have the capacity to differentiate into both neurons and glial cells (Acosta et al., 2009),

GATA3 may closely regulate the differentiation of BE(2)-C cells and determine the differentiation status of neuroblastoma stem cells.

To investigate the effects of GATA3 on neuronal differentiation in BE(2)-C cells, the expression of a group of neuronal markers is investigated, including TH, peripherin,

SNAP25, Rab5A and Rab1B.

Tyrosine hydroxylase (TH) is a specific marker for sympathetic neurons because it is the first and rate-limiting enzyme in synthesizing the catecholamine neurotransmitters.

In the rat embryonic study, TH is started to express in the cells of thoracic sympathetic ganglia on E11, and occurs in abdominal and lumbar ganglia on E12~13

(Teitelman et al., 1979). Embryonic mouse superior cervical ganglia show a similar

developmental time course in the neuronal differentiation by TH detection (Coughlin

et al., 1977). In BE(2)-C cells, GATA3 overexpression induces the upregulation of

TH expression (Figure 6B), which is consistent with the role of GATA3 in the

sympathetic development whereas the alteration of TH expression is not significant in

the GATA3 deficiency context.

SNAP25 is a palmitoylated protein which assembles with syntaxin as a key component of the “t-SNARE” complex and locates at the synaptic plasma membrane

(Chapman et al., 1994; McMahon and Sudhof, 1995). Proteins of the synaptic vesicle

membrane (such as synaptobrevin, synaptophysin, synapsin and synaptotagmin)

interact with their plasma membrane-located counterparts (such as SNAP25 and

syntaxin) to facilitate synaptic vesicle docking and calcium dependent exocytosis

(Kretzschmar et al., 1996). The expression of these vesicle exocytosis regulators is an

important characteristic of neuronal cells. For example, postmitotic NT2N cells,

which are considered to be a good in vitro model for mature neurons in the human

central nervous system (CNS), are derived from human NT2 teratocarcinoma cells by

a retinoic acid (RA) treatment. The vesicle exocytosis proteins are dramatically

expressed in NT2N cells and can’t be detected in untreated NT2 cells or cells exposed

to RA for only 6 days (Sheridan and Maltese, 1998). Based on these facts, the

phenomena that the SNAP25 expression is decreased under GATA3 deficiency and is

increased during GATA3 overexpression in our experiments (Figure 6B) provide

solid support for the role of GATA3 in the induction of neuronal differentiation in

BE(2)-C cells.

Peripherin is a peripheral neuronal marker which is a type of III intermediate filament

(IF) protein mainly found in the neurons of the peripheral nervous system (PNS) such

as motor, sensory and sympathetic neurons as well as neuroendocrine carcinomas of

the skin and certain melanomas (Baudoin et al., 1993; Huttenbach et al., 2002; Portier

et al., 1983). The fact that peripherin expression remains constant in both the GATA3 100

deficiency and overexpression context (Figure 6B) is consistent with the previous

study that loss of GATA3 doesn’t influence generic neuronal properties (Moriguchi et al., 2006).

Rab proteins are a family of Ras-related GTPases that regulate vesicular traffic in

mammalian cells (Novick and Zerial, 1997). Around 70 different Rab proteins have

been identified in humans so far. The different Rab GTPases are localized to the

cytosolic face of specific intracellular membranes, where they regulate multiple steps

of membrane traffic, including vesicle formation, vesicle movement along actin and

tubulin networks and membrane fusion. These processes constitute the route through

which cell surface proteins are trafficked from the Golgi to the plasma membrane and

are recycled (Stenmark and Olkkonen, 2001). Rab5A is related to a active status of

the endocytic pathway in recycling of synaptic vesicles in neurons (Sudhof, 1995).

The expression of Rab5A in differentiated NT2N cells is 4 to 5 fold higher than in

undifferentiated NT2 cells (Sheridan and Maltese, 1998). However, in BE(2)-C cells,

the expression level of Rab5A is not influenced by GATA3 expression (Figure 6B),

which indicates that Rab5A is not a suitable neuronal marker in this system. RablB

functions in constitutive transport of proteins between the endoplasmic reticulum and

Golgi apparatus (Plutner et al., 1991) and may relate to the cell proliferative status. In

our results, upregulation of Rab1B under loss of GATA3 is related to a higher

proliferative status, whereas downregulation of Rab1B during GATA3 overexpression

is committed to a decreased proliferative status (Figure 6B), which further confirms

the role of GATA3 in the regulation of the BE(2)-C cell proliferation (Figure 1 to 4).

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Two glial cell markers, GFAP and vimentin, are investigated to confirm the role of

GATA3 in differentiation. GFAP and vimentin both belong to Class III family of IF proteins and distribute in diverse glial cells. The GFAP is specifically expressed in astrocytes in the CNS; in the PNS, it has been detected in the satellite cells and non- myelinating Schwann cells (Sancho-Tello et al., 1995; Stephenson and Byers, 1995;

Triolo et al., 2006). Neural stem cells also strongly express GFAP (Imura et al., 2003).

Vimentin is also expressed in both early and mature glial cells in the nervous system

(Schnitzer et al., 1981). In BE(2)-C cells, increased GFAP expression is shown under loss of GATA3, while decreased GFAP expression is induced by GATA3 overexpression (Figure 6C), which is consistent with the role of GATA3 in the neuronal differention in BE(2)-C cells.

In summary, GATA3 deficiency induces the glial differentiation while GATA3 itself promotes the neuronal differentiation in BE(2)-C cells. Interestingly, we found that

GATA3 expression is not shown in the typical S-type neuroblastoma cell line -

SHEP1, but is highly exhibited in I-type and most N-type cell lines (Figure 7A). This result further confirms the role of GATA3 in the neuroblastoma differentiation.

SHEP1 cells can represent an extreme model of GATA3 deficiency. However, we also observed that GATA3 is downregulated during the RA-treated neuronal differentiation (Figure 7B). In the RA treatment time course study, GATA3 expression starts to dramatically decrease after 3 days, thereafter, the expression can’t be detected. The RA-induced strong neuronal differentiation in BE(2)-C cells as is shown in Figure 7C. The neuronal differentiation also begins after 3-day treatment 102

and progresses in the differentiation status thereafter until fully differentiated. So the

pattern of GATA3 downregulation is consistent with the neuronal differentiation

process in the time course. These observations suggest that GATA3 plays an

important role in neuronal progenitors. If cells exist in a terminal differentiation status,

GATA3 expression is not expected.

Finally, a progenitor cell marker nestin was investigated in BE(2)-C cells. Nestin is

one kind of IF protein (Hockfield and McKay, 1985). Nestin was first identified as a

specifically expressed gene in neuroepithelial stem cells and the nestin expression

distinguishes stem cells from more differentiated cells in the neural tube (Lendahl et

al., 1990). Later, it was found that nestin is widely distributed in most neuronal

progenitor cells in the CNS (Dahlstrand et al., 1995), in migrating neural crest cells

(Lothian and Lendahl, 1997; Stemple and Anderson, 1992) and in adult radial glial

cells (Chanas-Sacre et al., 2000). Following transition to differentiated cells, nestin

expression decreases and is substituted by other tissue-specific IFs (Dahlstrand et al.,

1995). In the PNS, nestin is expressed in both neuronal and glial progenitor cells (Shi

et al., 2008). In BE(2)-C cells, nestin is highly expressed but its expression is not

affected by the alteration of GATA3 expression (Figure 6D). This suggests that

BE(2)-C cells, as a kind of neuroblastom stem cells, are still in a progenitor status.

The modulation between GATA3 and other sympathetic development regulators

GATA3 was first identified as a gene specifically required for the expression of

adrenergic traits because in GATA3 mutant mice, sympathetic ganglia form and

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express pan-neuronal markers and Phox2 proteins, but fail to express TH and DBH

(Lim et al., 2000). GATA3 is genetically downstream of Phox2b because its

expression in sympathetic precursor cells is abolished in Phox2b mutants (Tsarovina

et al., 2004).

In 2006, Moriguchi et al. found that the expression levels of Mash1, dHand and

Phox2b (postulated upstream regulators of GATA3) as well as downstream products,

TH and DBH, are markedly downregulated in the developing sympathoadrenal

system of GATA3 mutant mice. In the transgenic mouse model that specifically

expresses GATA3 in the sympathoadrenal lineages under the human DBH promoter, the restoration of GATA3 recovers the sympathoadrenal function almost to the normal level and phenotypically restores the downstream as well as the putative

upstream genes, which suggests that the sympathoadrenal differentiation is controlled

by mutually reinforcing feedback transcriptional modulations between GATA3,

Mash1, dHand and Phox2b (Moriguchi et al., 2006).

However, there were no in vitro studies performed before to confirm the modulation

between GATA3 and other sympathetic regulators. In our study, the direct

modulations between GATA3 and Mash1, GATA3 and Phox2b were investigated in

BE(2)-C cells under the GATA3 knockdown or overexpression context. It is

demonstrated that Mash1 is significantly upregulated in GATA3 overexpression cells,

while Phox2b is significantly downregulated in GATA3 knockdown cells (Figure 8).

These results provide the direct evidence that GATA3 directly regulates the

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expression of Mash1 and Phox2b at the cellular level. Therefore, these postulated upstream genes actually have a bi-directional modulation with GATA3.

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Discussion

To investigate the role of GATA3 in neuroblastoma stem cells, it is important to learn

the GATA3 function in the normal development of the sympathetic nervous system as

well as other tissues. In the normal sympathetic development, loss of GATA3 induces

embryonic lethality around E11 due to noradrenaline deficiency and leads to increased apoptosis and significant impairment of sympathetic differentiation. The size of sympathetic ganglia is dramatically reduced in GATA3-/- mice due to a strong

increase in the number of apoptotic cells (Tsarovina et al., 2004). However, in the development of the kidney, nephric duct-specific inactivation of GATA3 leads to

massive ectopic ureter budding and premature nephric duct cell differentiation which suggests that loss of GATA3 promotes both proliferation and differentiation of

nephric progenitor cells (Grote et al., 2008). In the development of breast epithelial

cells, GATA3 deficiency leads to an expansion of luminal progenitors and a

concomitant block in differentiation, while introduction of GATA3 into a stem cell-

enriched population induces maturation along the alveolar luminal lineage (Asselin-

Labat et al., 2007). Based on the above phenomena, the effects of GATA3 loss in the

nephric development are completely opposite to those in the sympathetic development,

whereas in the mammary development GATA3 deficiency induces proliferation as in

nephric ducts and blocks differentiation as in the sympathetic system. Therefore,

though GATA3 plays essential roles in the mammalian development in multiple

organs, the underlying mechanisms are diverse. But the differences are presented in

the cellular level; in the organic level, loss of GATA3 consistently results in developmental defects, including shrinkage of sympathetic ganglia, a spectrum of

106

urogenital malformations and the mammary undifferentiated luminal cell expansion

with basement-membrane detachment that leads to the caspase-mediated cell death in

the long term. In summary, GATA3 is active in stem cells or progenitor cells in

different organs and contributes to the normal mammalian development.

In our I-type neuroblastoma cell system, loss of GATA3 results in increased

proliferation potential and blocking commitment to neuronal differentiation.

Compared with the normal sympathetic development, GATA3 shows a similar

function in differentiation but has opposite effects on proliferation. The conflict in the

effects on proliferation may reflect the difference between in vivo and in vitro studies.

The sympathetic proliferation in vivo is not only influenced by the local loss of

GATA3 but also controlled by more integral factors such as defects from other organs due to GATA3 deficiency. The defects in the kidney, heart, thymus and hematopoietic

system et al. would result in an abnormal status in the whole body and further have

effects on local organs. The apoptosis in the sympathetic ganglia and adrenal medulla

might be induced by some specific factors produced in the abnormal development, but

not by the direct loss of GATA3. Another possibility is that GATA3 is indispensible

in maintaining the neuronal progenitor cell survival. If GATA3 expression is deleted,

these neuronal progenitor cells can’t survive and apoptosis is induced. This possibility

can be proven by deleting the GATA3 expression in N-type neuroblastoma cell lines,

such as SK-N-DZ or SK-N-AS et al. If apoptosis is induced in these N-type

neuroblastoma cell lines, this possibility might be true. In our BE(2)-C cell system,

the external factors are eliminated, so the increased proliferation induced by GATA3

deficiency might reflect the direct effect on the sympathetic development. However,

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the BE(2)-C cell line belongs to the intermediate I-type neuroblastoma stem cells, so its situation might be different from N-type neuroblastoma cell lines. Based on observations, GATA3 deficiency induces increased proliferation in SK-N-AS cells, but leads to apoptosis in SK-N-DZ cells. Therefore, even in N-type neuroblastoma cell lines, the effects of GATA3 loss are diverse; this might be derived from the

delicate differentiated status of N-type cells.

Previous studies demonstrated that breast cancers might originate from breast cancer

stem cells (Dontu, 2008). Similarly, loss of GATA3 function contributes to the

tumorigenesis of breast cancers (Usary et al., 2004). Loss of GATA3 marks a

progression from adenoma to early carcinoma and the onset of tumor dissemination in

breast cancers. Restoration of GATA3 in late carcinomas induces tumor

differentiation and suppresses tumor dissemination. Targeted deletion of GATA3 in

early tumors leads to apoptosis of differentiated cells, which suggests that the loss of

GATA3 is not sufficient for malignant conversion, while an expanding GATA3-

negative tumor cell population accompanies malignant progression (Kouros-Mehr et

al., 2008).

In summary, loss of GATA3 in cancer stem cells promotes tumor progression. The

expression level of GATA3 in cancer stem cells can be a good prognosis marker of tumors. Low expression of GATA3 in tumors might predict a more progressive and malignant tendency in the tumor development. The prediction would help to start

suitable treatments in the early stage of tumor growth.

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Amplification or overexpression of Cyclin D1 is important in tumorigenesis in

multiple organs including the parathyroid adenoma, lymphoma, breast and prostate

cancers (de Boer et al., 1997; Drobnjak et al., 2000; Hsi et al., 1996; Musgrove et al.,

1994). Recently, Dr. Richard Pestell and colleagues at Thomas Jefferson University found that Cyclin D1 is required for the breast cancer growth in mice. The therapy targeting Cyclin D1 may block the expansion of breast cancer stem cells and effectively control tumor growth. Amplication of the Cyclin D1 gene has been also

found in some neuroblastoma tumors (Molenaar et al., 2003). In our study, the role of

GATA3 in the increasing clonogenic capacity of neuroblastoma stem cells might

through the downstream Cyclin D1 gene, which suggests that targeting Cyclin D1 in

the therapy might be an effective approach to decreasing the proliferative potential of

neuroblastoma stem cells and effectively cure this cancer.

Cyclin D1 promotes cell cycle progression via the typical Cyclin D1-pRb-E2F

pathway. The active E2F factors become detached from pRb and induce the synthesis

of transcription factors required for entering the S phase. However, in our study the

significant downregulation of E2F1 expression accompanied by the increased

proliferation suggests that there must be another pathway to promote proliferation by

reduced E2F1 in addition to the typical Cyclin D1-pRb-E2F pathway. Actually, the role of E2F1 in tumorigenesis is complex and not completely clear. In addition to

promoting proliferation, E2F1 can also potently induce apoptosis when it is

overexpressed or deregulated through pRb inactivation (Tsai et al., 1998; Wu and

Levine, 1994). Various mouse models have been reported to show an oncogenic or 109

tumor suppressive property for E2F1. Transgenic mice overexpressing E2F1 in

squamous epithelial tissues or in the liver are predisposed to developing tumors

(Conner et al., 2000; Pierce et al., 1998; Pierce et al., 1999). On the other hand, mice

lacking a functional E2F1 allele are also predisposed to the tumor development and in

this case E2F1 may inhibit tumorigenesis by promoting apoptosis (Yamasaki et al.,

1996). Our research implies that the primary function of E2F1 in this I-type

neuroblastoma stem cell system is to induce apoptosis, but not promote proliferation.

E2F1 downregulation induced by loss of GATA3 results in blocking apoptosis and

contributes to increased clonogenicity. This second pathway might expand the effects

of GATA3 deficiency on proliferation (Figure 9).

In differentiation studies, GATA3 promotes the neuronal differentiation in BE(2)-C

cells, which is consistent with the GATA3 function in the normal sympathetic

development. Interestingly, loss of GATA3 not only blocks the commitment to

neuronal differentiation, but also promotes glial differentiation in BE(2)-C cells as

evidenced by the GFAP upregulation. It is well known that the transcription factors

Mash1, Phox2b, Phox2a, dHand and GATA3 are necessary for sympathetic neuronal

differentiation, but there are no reports that they also have effects on glial

differentiation. One possibility is that loss of GATA3 induces apoptosis and masks

further effects in normal sympathetic ganglia or adrenal medulla. Since sympathetic

neurons and glial cells are both originated from NCSCs, it is possible that suppression

of GATA3 and other sympathetic neuronal regulators in glial progenitors is essential

for glial differentiation in the sympathetic nervous system. Lack of GATA3 expression in the typical S-type neuroblastoma cell line SHEP1 supports this point.

110

Although in 2006, Moriguchi et al. already demonstrated that GATA3 is not only a

downstream gene of Mash1, Phox2b and dHand but also possesses mutual

modulations with these sympathetic regulators (Moriguchi et al., 2006), no in vitro

study has been investigated to prove this since then. In the mouse study, the reduced

expression of Mash1, Phox2b and dHand induced by GATA3 deficiency is

accompanied by the shrinkage of sympathetic ganglia or adrenal medulla due to

apoptosis, so it’s difficult to judge if the decreased expression is directly derived from

loss of GATA3 or other integral factors. From the data derived from BE(2)-C cells,

we can confirm that the modulations between GATA3 and Mash1, GATA3 and

Phox2b are directly at the cellular level. In addition, the Phox2b and Mash1 promoters

have multiple (A/T)GATA(A/G) sequences, which provides the possibility that

GATA3 might bind to their promoters to promote transcription.

The cancer stem cell model demonstrates the origin of tumorigenesis. Only if therapies targeting killing cancer stem cells are employed, tumors can be eliminated, or else new tumors will recur repeatedly. Therefore, it’s important to investigate the physiology of cancer stem cells. In this study, we used BE(2)-C cells as a neuroblastoma stem cell model and investigated the GATA3 regulation in this system.

The mechanisms underlying enhanced clonogenicity induced by GATA3 deficiency provide more information for new neuroblastoma therapies.

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Materials and methods

Cell culture

The human neuroblastoma I-type BE(2)-C cells (ATCC CRL-2268) were maintained

in Dulbecco’s modified Eagle’s medium (DMEM) / F12 (1:1) with L-Glutamine

(Hyclone) supplemented with 10% fetal bovine serum (FBS) (Atlantic Biologicals)

and 1% penicillin/streptomycin (Fisher) at 37°C in a humidified 5% CO2 incubator.

BE(2)-C cells were subcultured at the split ratio of 1:3 every 3 days.

The 293TN cells (a gift from Dr. William Maltese), a pseudoviral particle packaging

cell line, were cultured in DMEM with 2% heat-inactivated FBS. The 293GPG human

kidney retroviral packaging cells (a gift from Dr. Richard Mulligan) were grown in

293 GPG medium which contains DMEM supplemented with

G418/puromycin/doxycyclin (Invitrogen), penicillin/streptomycin, and 10% heat-

inactivated FBS.

GATA3 knockdown in the BE(2)-C cell line

Construct

The lentiviral expression vector – pSIH1-H1-puro shRNA Expression Lentivector

(SBI) – was provided by Dr. William Maltese. A short hairpin RNA was designed to

target a 19 bp sequence specific to human GATA3 mRNA (Genebank NM

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001002295). The oligonucleotide sequence, 5’-352CCAAGACGTCCATCCACCA370-

3’, was designed by Oligoengine and synthesized by IDT. The sequence is followed

by a 12-nucleotide non-complementary spacer (CTTCCTGTCAGA) and the reverse

complement of the initial 19-nucleotide sequence. The sticky ends of the hairpin RNA

were specifically cloned into the BmHI and EcoRI sites in the pSIH1-H1-puro vector

to generate pSIH1-H1-puro-GATA3si. The pSIH1-H1-puro-GFPsi vector was used as

the control.

Infection

Lentivirus was produced in 293TN packaging cells. For transfection, the 293TN cells

were seeded at 3.75×106 per 100mm dish in DMEM containing 10% heat-inactivated

FBS. 24 hours later, the cells were transfected with the pSIH1-H1-puro- GATA3si

vector and three lentiviral packaging plasmids (pPack-Rev, pPack-Gag and pPack-

VSVG, provided by Dr. William Malese) by Lipofectamine (Invitrogen). After 24 hours, cells were started growing in DMEM containing 2% heat-inactivated FBS. 48

hours after transfection, the virus-enriched medium was collected and passed through

a 0.45µm filter to infect target cells (BE(2)-C cells) in the presence of 4µg/ml

polybrene (Sigma).

Selection

72 hours after infection, the medium was replaced on the infected BE(2)-C cells by

DMEM / F12 (1:1) with L-Glutamine. After being cultured for three days, 2µg/ml

puromycin (MP Biomedicals) was added to the medium and kept for 6 days. After 6- 113

day selection, the surviving cells were pooled and used for studies in the following

sections.

GATA3 overexpression in the BE(2)-C cell line

The pBabe-puro-GATA3 construct was obtained from Dr. Charles M. Perou. A

pBabe-puro vector was used as the control. 293GPG packaging cells were used to

produce retrovirus. For transfection, the 293GPG cells were seeded at 5×106 per

60mm dish in DMEM containing 10% heat-inactivated FBS. After 24 hours, the cells were transfected with the pBabe-puro-GATA3 or pBabe-puro vectors by

Liferfectamine. 5 hours later, cells were started growing in the 293GPG medium. 48

hours after transfection, the retroviral supernatant was harvested and passed through a

0.45 μm filter to infect BE(2)-C cells every 8 hours for 4 times in the presence of

4µg/ml polybrene. One day after the final round of infection, cells were cultured in the presence of 2.5µg/ml puromycin for 10 days, and drug-resistant cells were pooled and used for studies in the following sections.

Soft agar colony formation assay

1,000 BE(2)-C cells were suspended as single cells in DMEM supplemented with

10% FBS and 0.3% Noble agar (Sigma) and plated on 6-well plates containing a

solidified bottom layer (0.6% Noble agar in DMEM supplemented with 10% FBS).

o After 10-14 days of incubation at 37 C, viable clones were stained with 5 mg/ml 3-

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[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma). The

colonies were photographed and counted.

Clonogenic assay

For the clonogenic assay, cells were seeded at 1,000 per dish in 100-mm dishes. The

medium was replaced in the cultures every 3 days. Colony formation was assessed

after 14 days by washing the cultures with PBS, fixing the cells for 15 min in a mix

with 10% acetic acid and 10% methanol, and staining for 15 min with 0.4% crystal violet in 20% methanol. The colonies were photographed and counted.

MTT assay

The viability of cell populations in culture was quantified by metabolic activity assay,

measuring the conversion of MTT to the formazan derivative. The formazan

derivative was quantified in a continuous 7-day period by measuring its absorbance at

570 nm and 690 nm with a Spectra Max 384 Plus plate reader. The net absorbance

(OD570 minus OD690) is the MTT reading.

Cell growth

To assess cell growth, GATA3 knockdown or overexpression cells were seeded in 35

mm dishes at 1000 or 1500 cells per dish on the initial day respectively. On the day 3,

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6, 9, 12, 15 and 18, the cells were harvested from three parallel dishes and counted

with a Coulter Z-series particle counter (Beckman-Coulter Corporation).

Immunoblot

Cells were suspended in standard sodium dodecyl sulfate (SDS) sample buffer.

Protein concentrations were determined with a Bio-Rad protein assay kit, using

bovine serum albumin as a reference. 50µg of protein was separated on 8%, 10% or

12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were then probed with antibodies, visualized by enhanced chemiluminescence (ECL) and quantified with an Alpha Innotech FluorChem HD2 imaging system. The following primary antibodies and dilutions were used: mouse anti-GATA3, 1:100

(Santa Cruz); rabbit anti-p16, 1:2000 (Santa Cruz); mouse anti-E2F1, 1:200 (Santa

Cruz); rabbit anti-pRb, 1:1000 (Santa Cruz); rabbit anti-p21, 1:200 (Santa Cruz); rabbit anti-p27, 1:200 (Santa Cruz); mouse anti-p53, 1:50 (Calbiochem); mouse anti-

Cyclin A, 1:2000 (Santa Cruz); mouse anti-Cyclin B1, 1:200 (Santa Cruz); mouse anti-Cyclin D1, 1:200 (Santa Cruz); rabbit anti-Cyclin E, 1:200 (Santa Cruz); mouse

anti-TH, 1:10,000 (Sigma); rabbit anti-peripherin, 1:10,000 (Chemicon); mouse anti-

SNAP25, 1:2500 (Serotec); rabbit anti-Rab5A, 1:1000 (Santa Cruz); rabbit anti-

Rab1B, 1:2000 (Zymed); rabbit anti-GFAP, 1:2500 (DAKO); mouse anti-vimentin,

1:2000 (Santa Cruz); mouse anti-nestin, 1:1000 (BD); mouse anti-Mash1, 1:250 (BD);

rabbit anti-Phox2b, 1:2000 (Jabs lab); mouse anti-α-tubulin, 1:8000 (Sigma).

Horseradish peroxidase-conjugated anti-mouse or anti-rabbit was used as secondary

antibodies (ICN) at dilution 1:4000.

116

Differentiation analysis

For differentiation assays, retinoic acid (RA) was dissolved in Me2SO and 10 mM stock solutions were prepared. BE(2)-C cells were treated with 10 µM RA for 1, 3, 7,

10 and 14 days to induce neuronal differentiation. Phase contrast pictures were taken at each time point. BE(2)-C cells treated with Me2SO were used as a negative control.

Statistical Analysis

Values are represented as Mean ± SD. A Two-tailed Student’s t-test was performed for two groups (the control group versus the GATA3 overexpression or downregulation group). A P value 0.05 is considered statistically significant.

117

Acknowledgements

We thank Dr. William Maltese for providing techniques to establish the GATA3 knockdown cell line. We thank Dr. Jean Overmeyer and Ashley Young for the troubleshooting in the process of GATA3 knockdown cell line establishment. We thank Dr. William Maltese for providing the SNAP25, Rab5A and Rab1B primary antibodies and Dr. Roy Collaco for providing pRb and p27 primary antibodies. This work is supported by the National Cancer Institute grant CA124982 (H.-F.D.).

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References

Acosta, S., Lavarino, C., Paris, R., Garcia, I., de Torres, C., Rodriguez, E., Beleta, H.,

and Mora, J. (2009). Comprehensive characterization of neuroblastoma cell line

subtypes reveals bilineage potential similar to neural crest stem cells. BMC Dev Biol

9, 12.

Aprelikova, O., Xiong, Y., and Liu, E. T. (1995). Both p16 and p21 families of

cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin- dependent kinases by the CDK-activating kinase. J Biol Chem 270, 18195-18197.

Asselin-Labat, M. L., Sutherland, K. D., Barker, H., Thomas, R., Shackleton, M.,

Forrest, N. C., Hartley, L., Robb, L., Grosveld, F. G., van der Wees, J., et al. (2007).

Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 9, 201-209.

Baudoin, C., Meneguzzi, G., Portier, M. M., Demarchez, M., Bernerd, F., Pisani, A.,

and Ortonne, J. P. (1993). Peripherin, a neuronal intermediate protein, is stably

expressed by neuroendocrine carcinomas of the skin, their xenograft on nude mice,

and the corresponding primary cultures. Cancer Res 53, 1175-1181.

Brodeur, G. M. (2003). Neuroblastoma: biological insights into a clinical enigma. Nat

Rev Cancer 3, 203-216.

Castleberry, R. P. (1997). Neuroblastoma. Eur J Cancer 33, 1430-1437; discussion

1437-1438.

119

Chanas-Sacre, G., Rogister, B., Moonen, G., and Leprince, P. (2000). Radial glia

phenotype: origin, regulation, and transdifferentiation. J Neurosci Res 61, 357-363.

Chapman, E. R., An, S., Barton, N., and Jahn, R. (1994). SNAP-25, a t-SNARE which

binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J

Biol Chem 269, 27427-27432.

Chong, J. L., Tsai, S. Y., Sharma, N., Opavsky, R., Price, R., Wu, L., Fernandez, S.

A., and Leone, G. (2009). E2f3a and E2f3b contribute to the control of cell proliferation and mouse development. Mol Cell Biol 29, 414-424.

Collecchi, P., Santoni, T., Gnesi, E., Giuseppe Naccarato, A., Passoni, A., Rocchetta,

M., Danesi, R., and Bevilacqua, G. (2000). Cyclins of phases G1, S and G2/M are

overexpressed in aneuploid mammary carcinomas. Cytometry 42, 254-260.

Conner, E. A., Lemmer, E. R., Omori, M., Wirth, P. J., Factor, V. M., and

Thorgeirsson, S. S. (2000). Dual functions of E2F-1 in a transgenic mouse model of

liver carcinogenesis. Oncogene 19, 5054-5062.

Coughlin, M. D., Boyer, D. M., and Black, I. B. (1977). Embryologic development of

a mouse sympathetic ganglion in vivo and in vitro. Proc Natl Acad Sci U S A 74,

3438-3442.

Dahlstrand, J., Lardelli, M., and Lendahl, U. (1995). Nestin mRNA expression

correlates with the central nervous system progenitor cell state in many, but not all,

regions of developing central nervous system. Brain Res Dev Brain Res 84, 109-129.

de Boer, C. J., van Krieken, J. H., Schuuring, E., and Kluin, P. M. (1997). Bcl-

1/cyclin D1 in malignant lymphoma. Ann Oncol 8 Suppl 2, 109-117.

120

Diehl, J. A. (2002). Cycling to cancer with cyclin D1. Cancer Biol Ther 1, 226-231.

Dontu, G. (2008). Breast cancer stem cell markers - the rocky road to clinical

applications. Breast Cancer Res 10, 110.

Drobnjak, M., Osman, I., Scher, H. I., Fazzari, M., and Cordon-Cardo, C. (2000).

Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone.

Clin Cancer Res 6, 1891-1895.

Galderisi, U., Jori, F. P., and Giordano, A. (2003). Cell cycle regulation and neural

differentiation. Oncogene 22, 5208-5219.

Galizia, G., Ferraraccio, F., Lieto, E., Orditura, M., Castellano, P., Imperatore, V.,

Romano, C., Vollaro, M., Agostini, B., Pignatelli, C., and De Vita, F. (2004).

Prognostic value of p27, p53, and vascular endothelial growth factor in Dukes A and

B colon cancer patients undergoing potentially curative surgery. Dis Colon Rectum 47,

1904-1914.

Grote, D., Boualia, S. K., Souabni, A., Merkel, C., Chi, X., Costantini, F., Carroll, T.,

and Bouchard, M. (2008). Gata3 acts downstream of beta-catenin signaling to prevent

ectopic metanephric kidney induction. PLoS Genet 4, e1000316.

He, G., Siddik, Z. H., Huang, Z., Wang, R., Koomen, J., Kobayashi, R., Khokhar, A.

R., and Kuang, J. (2005). Induction of p21 by p53 following DNA damage inhibits

both Cdk4 and Cdk2 activities. Oncogene 24, 2929-2943.

Hockfield, S., and McKay, R. D. (1985). Identification of major cell classes in the

developing mammalian nervous system. J Neurosci 5, 3310-3328.

121

Hsi, E. D., Zukerberg, L. R., Yang, W. I., and Arnold, A. (1996). Cyclin D1/PRAD1 expression in parathyroid adenomas: an immunohistochemical study. J Clin

Endocrinol Metab 81, 1736-1739.

Huttenbach, Y., Prieto, V. G., and Reed, J. A. (2002). Desmoplastic and spindle cell melanomas express protein markers of the neural crest but not of later committed stages of Schwann cell differentiation. J Cutan Pathol 29, 562-568.

Imura, T., Kornblum, H. I., and Sofroniew, M. V. (2003). The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP. J Neurosci 23, 2824-2832.

Klopfleisch, R., and Gruber, A. D. (2009). Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands. Res Vet Sci.

Knudsen, K. E., Diehl, J. A., Haiman, C. A., and Knudsen, E. S. (2006). Cyclin D1: polymorphism, aberrant splicing and cancer risk. Oncogene 25, 1620-1628.

Koljonen, V., Tukiainen, E., Haglund, C., and Bohling, T. (2006). Cell cycle control by p21, p27 and p53 in Merkel cell carcinoma. Anticancer Res 26, 2209-2212.

Kouros-Mehr, H., Bechis, S. K., Slorach, E. M., Littlepage, L. E., Egeblad, M., Ewald,

A. J., Pai, S. Y., Ho, I. C., and Werb, Z. (2008). GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. Cancer Cell 13, 141-152.

Kretzschmar, S., Volknandt, W., and Zimmermann, H. (1996). Colocalization on the same synaptic vesicles of syntaxin and SNAP-25 with synaptic vesicle proteins: a re- evaluation of functional models required? Neurosci Res 26, 141-148.

122

Lendahl, U., Zimmerman, L. B., and McKay, R. D. (1990). CNS stem cells express a new class of intermediate filament protein. Cell 60, 585-595.

Lillevali, K., Haugas, M., Matilainen, T., Pussinen, C., Karis, A., and Salminen, M.

(2006). Gata3 is required for early morphogenesis and Fgf10 expression during otic

development. Mech Dev 123, 415-429.

Lim, K. C., Lakshmanan, G., Crawford, S. E., Gu, Y., Grosveld, F., and Engel, J. D.

(2000). Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the

sympathetic nervous system. Nat Genet 25, 209-212.

Lotayef, M., Wilson, G. D., Daley, F. M., Awwad, H. K., and Shouman, T. (2000).

Aberrant expression of cyclin A in head and neck cancer. Br J Cancer 83, 30-34.

Lothian, C., and Lendahl, U. (1997). An evolutionarily conserved region in the second

intron of the human nestin gene directs gene expression to CNS progenitor cells and

to early neural crest cells. Eur J Neurosci 9, 452-462.

McMahon, H. T., and Sudhof, T. C. (1995). Synaptic core complex of synaptobrevin,

syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem 270,

2213-2217.

Minegishi, N., Morita, S., Minegishi, M., Tsuchiya, S., Konno, T., Hayashi, N., and

Yamamoto, M. (1997). Expression of GATA transcription factors in myelogenous

and lymphoblastic leukemia cells. Int J Hematol 65, 239-249.

Molenaar, J. J., van Sluis, P., Boon, K., Versteeg, R., and Caron, H. N. (2003).

Rearrangements and increased expression of cyclin D1 (CCND1) in neuroblastoma.

Genes Chromosomes Cancer 36, 242-249.

123

Moriguchi, T., Takako, N., Hamada, M., Maeda, A., Fujioka, Y., Kuroha, T., Huber,

R. E., Hasegawa, S. L., Rao, A., Yamamoto, M., et al. (2006). Gata3 participates in a

complex transcriptional feedback network to regulate sympathoadrenal differentiation.

Development 133, 3871-3881.

Musgrove, E. A., Lee, C. S., Buckley, M. F., and Sutherland, R. L. (1994). Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle. Proc Natl Acad Sci U S A 91, 8022-8026.

Nakagawara, A. (1998). Molecular basis of spontaneous regression of neuroblastoma:

role of neurotrophic signals and genetic abnormalities. Hum Cell 11, 115-124.

Nawijn, M. C., Ferreira, R., Dingjan, G. M., Kahre, O., Drabek, D., Karis, A.,

Grosveld, F., and Hendriks, R. W. (2001). Enforced expression of GATA-3 during T

cell development inhibits maturation of CD8 single-positive cells and induces thymic

lymphoma in transgenic mice. J Immunol 167, 715-723.

Novick, P., and Zerial, M. (1997). The diversity of Rab proteins in vesicle transport.

Curr Opin Cell Biol 9, 496-504.

Patient, R. K., and McGhee, J. D. (2002). The GATA family (vertebrates and

invertebrates). Curr Opin Genet Dev 12, 416-422.

Pierce, A. M., Gimenez-Conti, I. B., Schneider-Broussard, R., Martinez, L. A., Conti,

C. J., and Johnson, D. G. (1998). Increased E2F1 activity induces skin tumors in mice heterozygous and nullizygous for p53. Proc Natl Acad Sci U S A 95, 8858-8863.

124

Pierce, A. M., Schneider-Broussard, R., Gimenez-Conti, I. B., Russell, J. L., Conti, C.

J., and Johnson, D. G. (1999). E2F1 has both oncogenic and tumor-suppressive

properties in a transgenic model. Mol Cell Biol 19, 6408-6414.

Plutner, H., Cox, A. D., Pind, S., Khosravi-Far, R., Bourne, J. R., Schwaninger, R.,

Der, C. J., and Balch, W. E. (1991). Rab1b regulates vesicular transport between the

endoplasmic reticulum and successive Golgi compartments. J Cell Biol 115, 31-43.

Portier, M. M., de Nechaud, B., and Gros, F. (1983). Peripherin, a new member of the

intermediate filament protein family. Dev Neurosci 6, 335-344.

Ross, R. A., Biedler, J. L., and Spengler, B. A. (2003). A role for distinct cell types in

determining malignancy in human neuroblastoma cell lines and tumors. Cancer Lett

197, 35-39.

Ross, R. A., Hein, A. M., Braca, J. A., 3rd, Spengler, B. A., Biedler, J. L., and

Scammell, J. G. (2002). Glucocorticoids induce neuroendocrine cell differentiation and increase expression of N-myc in N-type human neuroblastoma cells. Oncol Res

13, 87-94.

Ross, R. A., and Spengler, B. A. (2007). Human neuroblastoma stem cells. Semin

Cancer Biol 17, 241-247.

Sancho-Tello, M., Valles, S., Montoliu, C., Renau-Piqueras, J., and Guerri, C. (1995).

Developmental pattern of GFAP and vimentin gene expression in rat brain and in radial glial cultures. Glia 15, 157-166.

Schildkraut, J. M., Moorman, P. G., Bland, A. E., Halabi, S., Calingaert, B., Whitaker,

R., Lee, P. S., Elkins-Williams, T., Bentley, R. C., Marks, J. R., and Berchuck, A.

125

(2008). Cyclin E overexpression in epithelial ovarian cancer characterizes an etiologic

subgroup. Cancer Epidemiol Biomarkers Prev 17, 585-593.

Schnitzer, J., Franke, W. W., and Schachner, M. (1981). Immunocytochemical

demonstration of vimentin in astrocytes and ependymal cells of developing and adult

mouse nervous system. J Cell Biol 90, 435-447.

Schwab, M., Westermann, F., Hero, B., and Berthold, F. (2003). Neuroblastoma:

biology and molecular and chromosomal pathology. Lancet Oncol 4, 472-480.

Sheridan, K. M., and Maltese, W. A. (1998). Expression of Rab3A GTPase and other

synaptic proteins is induced in differentiated NT2N neurons. J Mol Neurosci 10, 121-

128.

Shi, H., Cui, H., Alam, G., Gunning, W. T., Nestor, A., Giovannucci, D., Zhang, M.,

and Ding, H. F. (2008). Nestin expression defines both glial and neuronal progenitors in postnatal sympathetic ganglia. J Comp Neurol 508, 867-878.

Slack, R., Lach, B., Gregor, A., al-Mazidi, H., and Proulx, P. (1992). Retinoic acid-

and staurosporine-induced bidirectional differentiation of human neuroblastoma cell

lines. Exp Cell Res 202, 17-27.

Smith, E., Hargrave, M., Yamada, T., Begley, C. G., and Little, M. H. (2002).

Coexpression of SCL and GATA3 in the V2 interneurons of the developing mouse

spinal cord. Dev Dyn 224, 231-237.

Spengler, B. A., Lazarova, D. L., Ross, R. A., and Biedler, J. L. (1997). Cell lineage

and differentiation state are primary determinants of MYCN gene expression and

malignant potential in human neuroblastoma cells. Oncol Res 9, 467-476.

126

Spirin, K. S., Simpson, J. F., Takeuchi, S., Kawamata, N., Miller, C. W., and Koeffler,

H. P. (1996). p27/Kip1 mutation found in breast cancer. Cancer Res 56, 2400-2404.

Stemple, D. L., and Anderson, D. J. (1992). Isolation of a stem cell for neurons and

glia from the mammalian neural crest. Cell 71, 973-985.

Stenmark, H., and Olkkonen, V. M. (2001). The Rab GTPase family. Genome Biol 2,

REVIEWS3007.

Stephenson, J. L., and Byers, M. R. (1995). GFAP immunoreactivity in trigeminal

ganglion satellite cells after tooth injury in rats. Exp Neurol 131, 11-22.

Sudhof, T. C. (1995). The synaptic vesicle cycle: a cascade of protein-protein

interactions. Nature 375, 645-653.

Sugimoto, T., Mine, H., Horii, Y., Takahashi, K., Nagai, R., Morishita, R., Komada,

M., Asada, Y., and Sawada, T. (2000). Neuroblastoma cell lines showing smooth

muscle cell phenotypes. Diagn Mol Pathol 9, 221-228.

Takeuchi, S., Koeffler, H. P., Hinton, D. R., Miyoshi, I., Melmed, S., and Shimon, I.

(1998). Mutation and expression analysis of the cyclin-dependent kinase inhibitor

gene p27/Kip1 in pituitary tumors. J Endocrinol 157, 337-341.

Teitelman, G., Baker, H., Joh, T. H., and Reis, D. J. (1979). Appearance of catecholamine-synthesizing enzymes during development of rat sympathetic nervous system: possible role of tissue environment. Proc Natl Acad Sci U S A 76, 509-513.

Thomson, S. P., and Meyskens, F. L., Jr. (1982). Method for measurement of self-

renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. Cancer Res 42, 4606-4613.

127

Tong, Q., Tsai, J., Tan, G., Dalgin, G., and Hotamisligil, G. S. (2005). Interaction between GATA and the C/EBP family of transcription factors is critical in GATA- mediated suppression of adipocyte differentiation. Mol Cell Biol 25, 706-715.

Toyoshima, H., and Hunter, T. (1994). p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78, 67-74.

Triolo, D., Dina, G., Lorenzetti, I., Malaguti, M., Morana, P., Del Carro, U., Comi, G.,

Messing, A., Quattrini, A., and Previtali, S. C. (2006). Loss of glial fibrillary acidic protein (GFAP) impairs Schwann cell proliferation and delays nerve regeneration after damage. J Cell Sci 119, 3981-3993.

Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L., and Jacks, T. (1998).

Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends survival of Rb-deficient mouse embryos. Mol Cell 2, 293-304.

Tsarovina, K., Pattyn, A., Stubbusch, J., Muller, F., van der Wees, J., Schneider, C.,

Brunet, J. F., and Rohrer, H. (2004). Essential role of Gata transcription factors in sympathetic neuron development. Development 131, 4775-4786.

Usary, J., Llaca, V., Karaca, G., Presswala, S., Karaca, M., He, X., Langerod, A.,

Karesen, R., Oh, D. S., Dressler, L. G., et al. (2004). Mutation of GATA3 in human breast tumors. Oncogene 23, 7669-7678. van Doorninck, J. H., van Der Wees, J., Karis, A., Goedknegt, E., Engel, J. D.,

Coesmans, M., Rutteman, M., Grosveld, F., and De Zeeuw, C. I. (1999). GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei. J

Neurosci 19, RC12.

128

Van Esch, H., Groenen, P., Nesbit, M. A., Schuffenhauer, S., Lichtner, P.,

Vanderlinden, G., Harding, B., Beetz, R., Bilous, R. W., Holdaway, I., et al. (2000).

GATA3 haplo-insufficiency causes human HDR syndrome. Nature 406, 419-422.

Walton, J. D., Kattan, D. R., Thomas, S. K., Spengler, B. A., Guo, H. F., Biedler, J. L.,

Cheung, N. K., and Ross, R. A. (2004). Characteristics of stem cells from human

neuroblastoma cell lines and in tumors. Neoplasia 6, 838-845.

Wang, A., Yoshimi, N., Ino, N., Tanaka, T., and Mori, H. (1997). Overexpression of

cyclin B1 in human colorectal cancers. J Cancer Res Clin Oncol 123, 124-127.

Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81,

323-330.

Wu, X., and Levine, A. J. (1994). p53 and E2F-1 cooperate to mediate apoptosis. Proc

Natl Acad Sci U S A 91, 3602-3606.

Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996).

Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85, 537-548.

Yamashita, M., Shinnakasu, R., Asou, H., Kimura, M., Hasegawa, A., Hashimoto, K.,

Hatano, N., Ogata, M., and Nakayama, T. (2005). Ras-ERK MAPK cascade regulates

GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. J

Biol Chem 280, 29409-29419.

129

Figure Legends

Figure 1 Soft agar colony formation assay for BE(2)-C cells with knockdown and overexpression of GATA3. (A) The pictures of the soft agar colony formation assay of BE(2)-C-pSIH1-H1-puro-GFPsi (abbr BE(2)-C-GFPsi) and BE(2)-C-pSIH1-H1- puro-GATAsi (abbr BE(2)-C-GATA3si) cell lines. 1000 individual BE(2)-C-GFPsi or

BE(2)-C-GATA3si cells were grown in soft agar for 11 days respectively. The clone number of BE(2)-C-GATA3si cells in soft agar is higher than that of BE(2)-C-GFPsi

cells. (B) The percentage of clones growing from 1000 cells in the BE(2)-C-GFPsi and BE(2)-C-GATA3si cell lines, represented as a mean ± SD (4 dishes). The

percentage of colony formation in BE(2)-C-GATA3si cells is significantly higher than

that of BE(2)-C-GFPsi cells (P<0.0001). (C) The pictures of the soft agar colony

formation assay of BE(2)-C-pBabe-puro and BE(2)-C-pBabe-puro-GATA3 cell lines.

1000 individual BE(2)-C-pBabe-puro or BE(2)-C-pBabe-puro-GATA3 cells were

grown in soft agar for 14 days respectively. The clone number of BE(2)-C-pBabe-

puro-GATA3 cells in soft agar is less than that of BE(2)-C-pBabe-puro cells. (D) The percentage of clones growing from 1000 cells in BE(2)-C-pBabe-puro and BE(2)-C- pBabe-puro-GATA3 cell lines, represented as a mean ± SD (4 dishes). The percentage of colony formation in BE(2)-C- pBabe-puro-GATA3 cells is significantly lower than that of BE(2)-C-pBabe-puro cells (P<0.0001).

Figure 2 Clonogenic assay for BE(2)-C cells with knockdown and overexpression of

GATA3. (A) The pictures of the clonogenic assay of BE(2)-C-GFPsi and BE(2)-C-

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GATA3si cell lines. 1000 individual BE(2)-C-GFPsi or BE(2)-C-GATA3si cells were

grown in 100 mm cell culture plates for 14 days respectively. The clone number of

BE(2)-C-GATA3si cells is higher than that of BE(2)-C-GFPsi cells. (B) The percentage of clones growing from 1000 cells in BE(2)-C-GFPsi and BE(2)-C-

GATA3si cell lines, represented as a mean ± SD (6 dishes). The percentage of colony

formation in BE(2)-C-GATA3si cells is significantly higher than that of BE(2)-C-

GFPsi cells (P<0.0001). (C) The pictures of the clonogenic assay of BE(2)-C-pBabe-

puro and BE(2)-C-pBabe-puro-GATA3 cell lines. 1000 individual BE(2)-C-pBabe-

puro or BE(2)-C-pBabe-puro-GATA3 cells were grown in 100 mm cell culture plates

for 14 days respectively. The clone number of BE(2)-C-pBabe-puro-GATA3 cells is

less than that of BE(2)-C-pBabe-puro cells. (D) The percentage of clones growing

from 1000 cells in BE(2)-C-pBabe-puro and BE(2)-C-pBabe-puro-GATA3 cell lines,

represented as a mean ± SD (6 dishes). The percentage of colony formation in BE(2)-

C- pBabe-puro-GATA3 cells is significantly lower than that of BE(2)-C-pBabe-puro

cells (P<0.0001).

Figure 3 MTT assays for BE(2)-C cells with knockdown and overexpression of

GATA3 in a continuous 7-day period. (A) MTT assays exhibit an increase in the number of viable cells in cultures of the BE(2)-C-GATA3si cell line in comparison with the BE(2)-C-GFPsi cell line. OD value is represented as a mean ± SD. Points, mean determined from six separate cultures in a 96-well plate initially seeded at 500 cells per well; bars, SD. (B) MTT assays exhibit a decrease in the number of viable

cells in cultures of the BE(2)-C- pBabe-puro-GATA3 cell line compared with the

BE(2)-C-pBabe-puro cell line. OD value is represented as a mean ± SD. Points, mean

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determined from four separate cultures in a 96-well plate initially seeded at 750 cells

per well; bars, SD. MTT was added directly to the medium to avoid losing detached

cells.

Figure 4 The growth rate of BE(2)-C cells with knockdown and overexpression of

GATA3. (A) BE(2)-C-GATA3si cells exhibit a marked increase in the growth rate compared with the control group of BE(2)-C-GFPsi cells. Cells were seeded in 35 mm

dishes on day 0 at an initial density of 1000 cells per dish. On each of the indicated

days, cells were harvested from three dishes in each group and counted with a Coulter

Z1 particle counter (mean ± SD). (B) BE(2)-C- pBabe-puro-GATA3 cells show a

dramatic decrease in the growth rate in comparison with the control group of BE(2)-

C-pBabe-puro cells. Cells were seeded in 35 mm dishes on day 0 at an initial density

of 1500 cells per dish. On each of the indicated days, cells were harvested from three

dishes in each group and counted with a Coulter Z1 particle counter (mean ± SD).

Figure 5 Immunoblot analyses of the expression of indicated cell cycle regulation

genes in BE(2)-C-GFPsi and BE(2)-C-GATA3si cell lines by representative samples.

P value is indicated on the left of each panel. P value is calculated from the

normalized units (gene expression level/loading control level) between the two groups.

Each group contains 3 to 4 samples and represented as a mean ± SD. (A) The

GATA3 expression is significantly decreased in GATA3 knockdown cells (P<0.05).

The level of α-tubulin is shown as the loading control. (B) The expression levels of p21, p27 and p53 in BE(2)-C-GATA3si cells show no significant difference from that

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of BE(2)-C-GFPsi cells. (C) The expression levels of p16, E2F1 and pRb in BE(2)-

C-GFPsi and BE(2)-C-GATA3si cells. The E2F1 expression is significantly decreased

(P<0.05) compared with that of the control, whereas the pRb expression is

significantly increased (P<0.05). (D) The expression levels of Cyclin A, B1, D1 and E in BE(2)-C-GFPsi and BE(2)-C-GATA3si cells. Cyclin B1 and Cyclin E are expressed equally in the control and the GATA3 knockdown cell line. The Cyclin D1 expression is significantly upregulated (P<0.05) compared with that of the control, whereas the Cyclin A expression is significantly downregulated (P<0.05).

Figure 6 Immunoblot analyses of the expression of indicated differentiation marker

genes in GATA3 knockdown and overexpression cell lines by representative samples.

P value is indicated on the left of each panel. P value is calculated from the

normalized units (gene expression level/loading control level) between the two groups.

Each group contains 3 to 4 samples and represented as a mean ± SD. (A) The

GATA3 expression is significantly decreased in GATA3 knockdown cells and increased in GATA3 overexpression cells (P<0.05). The level of α-tubulin is shown as the loading control. (B) The expression levels of neuronal marker genes and Rab

GTPase genes are presented in GATA3 knockdown and overexpression cell lines. The

TH expression is significantly upregulated in the GATA3 overexpression cell line in comparison with its control (P<0.05), whereas there is no significant difference of the

TH expression between the GATA3 knockdown cell line and its control. The

SNAP25 expression is significantly decreased in GATA3 knockdown cells (P<0.05)

while it is significantly increased in GATA3 overexpression cells (P<0.05). One of the Rab GTPase genes, Rab 1B, shows a significant increase in GATA3 knockdown

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cells (P<0.05) while it exhibits a significant decrease in GATA3 overexpression cells

(P<0.05). Peripherin and Rab5A are expressed equally in four lanes. (C) The

expression of glial marker genes in GATA3 knockdown and overexpression cell lines.

The GFAP expression is significantly upregulated in GATA3 knockdown cells

(P<0.05) and significantly downregulated in GATA3 overexpression cells (P<0.05).

Vimentin shows no difference of expression levels in four lanes. (D) The expression

of the progenitor cell marker gene, nestin, shows equal expression in GATA3

knockdown and overexpression cell lines.

Figure 7 Immunoblot analyses of GATA3 expression levels in different

neuroblastoma cell lines and the retinoic acid (RA) – treated BE(2)-C cell line. (A)

The expression of GATA3 in 11 neuroblastoma cell lines, including the I-type cell line, BE(2)-C; the S-type cell line SHEP1; others are N-type cell lines. The α-tubulin

levels are shown as the loading control. (B) The time course study of GATA3

expression in RA-treated BE(2)-C cells. Cells were collected on day 1, 3, 7, 10, 14 after RA treatment parallel with untreated controls at same time points. (C) The

morphology of BE(2)-C cells on day 1, 3 and 14 after RA treatment.

Figure 8 Immunoblot analyses of the expression of indicated sympathetic

development regulator genes in GATA3 knockdown and overexpression cell lines by

representative samples. P value is indicated on the left of each panel. P value is

calculated from the normalized units (gene expression level/loading control level)

between the two groups. Each group contains 3 to 4 samples and represented as a

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mean ± SD. The Mash1 expression shows a significant increase in the GATA3

overexpression cell line (P<0.05). In the GATA3 knockdown cell line, even though

the Mash1 expression exhibits an inclination to decrease, the difference is

insignificant compared with the control. In contrast, the Phox2b expression is

significantly downregulated in the GATA3 knockdown cell line (P<0.05), whereas the increase of its expression in the GATA3 overexpression cell line is insignificant in comparison with the control.

Figure 9 A simplified schematic diagram for the mechanisms underlying GATA

deficiency induced enhanced clonogenicity.

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SUMMARY/DISCUSSION

This dissertation includes two parts of work. In the first part, we investigated the postnatal development of mouse sympathetic ganglia and found that it is characterized predominantly by gliogenesis and a small amount of neurogenesis.

Nestin is identified as a molecular marker for both sympathetic glial and neuronal progenitors. In the second part, we demonstrated the role of a GATA family zinc finger transcription factor, GATA3, in the regulation of human neuroblastoma stem cell activities. We use BE(2)-C cells, a I-type neuroblastoma cell line, as the neuroblastoma stem cell model. We found that GATA3 deficiency induces increased clonogenicity in BE(2)-C cells through the upregulation of Cyclin D1 and the downregulation of E2F1 whereas overexpression of GATA3 results in decreased clonogenicity of BE(2)-C cells. In the differentiation study, we demonstrated that loss of GATA3 induces gliogenesis and overexpression of GATA3 promotes neurogenesis in BE(2)-C cells. GATA3 regulates differentiation in BE(2)-C cells through the modulations with the other sympathetic regulators, Mash1 and Phox2b, at the cellular level.

In the relationship, the first part of work is the basic research for the second part. A detailed characterization of the cellular basis for the postnatal sympathetic development facilitates the investigation of tumorigenesis in neuroblastoma. It is well established that neuroblastoma originates from the developing sympathetic nervous system. Based on the cancer stem cell model, neuroblastoma stem cells are the origin of neuroblastoma formation. Neuroblastoma stem cells are most likely derived from

146

transformed neural crest stem cells or sympathetic progenitor cells that obtain stem

cell properties through mutations.

Since 90% of children with this disease are diagnosed before 6 years old, there

are two possible phases in which neuroblastoma stem cells can be generated – the

embryonic and postnatal developmental phase. In the embryonic development,

sympathetic ganglia are composed of intensely proliferating neural crest stem cells,

neurons and glial cells, though their temporal developmental differences are dramatic.

Therefore, neuroblastoma stem cells can be derived from any type of cells in any time

during the embryonic sympathetic development due to genetic mutations. However, in

the postnatal sympathetic ganglia, the situation is different. During this phase, the

neural crest stem cells are either consumed or hibernating in the niche; the majority of

neurogenesis has completed and only a very small amount of neurons are proliferating;

gliogenesis continues after the embryonic development and dominates the postnatal

development, whereas it shows a gradually decreasing proliferating tendency and the

proliferation is completed by the adult phase. Therefore, the origin of neuroblastoma

stem cells in this phase is difficult to judge because the three types of cells are either

resting or going down in the proliferative ability. The cellular mutations or external

signals that can induce increased proliferation or gain of stem cell properties in the three cell types need to be investigated to explore the formation of neuroblastoma stem cells in the postnatal sympathetic development.

GATA3 deficiency has been demonstrated to increase clonogenicity in the I-

type neuroblastoma cell line, BE(2)-C. Loss of GATA3 might be a promoting factor

in the transformation of neural crest stem cells or sympathetic progenitor cells to form neuroblastoma stem cells. Previous studies indicated that GATA3 deficiency induces

147

a progenitor cell expansion in the kidney and mammary development (Asselin-Labat

et al., 2007; Grote et al., 2008). Loss of GATA3 promotes the dissemination and

metastasis in breast cancers (Kouros-Mehr et al., 2008). These facts suggest that

GATA3 deficiency might play a similar role in the neuroblastoma formation.

To prove this point, a series of experiments can be designed to investigate the

role of GATA3 in the tumorigenesis of neuroblastoma. For example, we can specifically knockout the GATA3 expression in the migrating neural crest stem cells, neuronal or glial progenitors during embryonic development, then observe if there is upregulated proliferation or transformed characteristics in these cells. This would

determine if loss of GATA3 is sufficient to induce tumorigenesis in the developing

sympathetic nervous system.

In this study, we also found Cyclin D1 and E2F1 are the downstream targets

of GATA3 in the proliferation regulation. Upregulation of Cyclin D1 and

downregulation of E2F1 are responsible for the increased proliferation induced by

GATA3 deficiency. We can further investigate if GATA3 can directly bind the

promoters of Cyclin D1 and E2F1 to regulate their expression. Are there any other

underlying mechanisms responsible for the increased clonogenicity induced by

GATA3 deficiency in BE(2)-C cells? Do the therapies targeting Cyclin D1 block the

expansion of neuroblastoma stem cells and effectively control the tumor growth?

It has been demonstrated in this research that GATA3 deficiency not only

suppresses neuronal differentiation, but also promotes glial differentiation. There are

no previous studies reporting that GATA3 plays a role in gliogenesis. GATA3 has

only been well established as a sympathetic neuronal regulator modulating with other

sympathetic neuronal regulators like Phox2b, Mash1, Phox2a, dHand. Our study 148

suggests that loss of GATA3 or other sympathetic neuronal regulators might also be

necessary for gliogenesis in the normal sympathetic development. The loss of GATA3 expression in the typical S-type neuroblastma cell line (similar to glial/Schwann cells)

– SHEP1 – has confirmed this point and suggests that SHEP1 cells might represent an

extreme status of GATA3 deficiency. It would be interesting to explore what are the

mechanisms of the suppressed GATA3 expression in the gliogenesis which expands

the GATA3 functions in the sympathetic development. GATA3 has been proven in

our study to modulate Phox2b and Mash1 at the cellular level in regulating the BE(2)-

C cell differentiation, it is of interest to investigate if GATA3 binds to their promoters

to regulate the expression at the genetic level?

Together with previous studies of the GATA3 function in the normal

sympathetic system and our data in I-type neuroblastma stem cells, we can determine

the role of GATA3 in the regulation of activities in the normal sympathetic

development and in the tumorigenesis of neuroblastoma, providing a good foundation

for future studies and clinical applications.

149

CONCLUSIONS

1. Nestin is a molecular marker for both sympathetic glial and neuronal progenitors in the mouse postnatal sympathetic development.

2. A population of nestin and BLBP double-positive cells in the mouse postnatal sympathetic ganglia is the direct precursors of sympathetic satellite cells. The mouse postnatal sympathetic development is characterized predominantly by gliogenesis and a very small amount of neurogenesis.

3. Our study reveals a temporal expression pattern of glial lineage markers that define the cellular intermediates in the process of satellite cell differentiation in mouse postnatal sympathetic development.

4. GATA3 deficiency induces increased clonogenicity of BE(2)-C cells, a type of

I-type neuroblastoma stem cell line, whereas GATA3 overexpression results in decreased clonogenicity of BE(2)-C cells.

5. The upregulation of Cyclin D1 and downregulation of E2F1 are possibly the downstream mechanisms that are responsible for the increased clonogenicity induced by GATA3 deficiency.

150

6. GATA3 deficiency induces glial differentiation in BE(2)-C cells whereas

GATA3 overexpression promotes neuronal differentiation.

7. GATA3 modulates with other sympathetic regulators Mash1 and Phox2b at the celluar level to regulate the BE(2)-C cell differentiation.

151

BIBLIOGRAPHY

Abbott, A. (2006). Cancer: the root of the problem. Nature 442, 742-743.

Abkowitz, J. L., Linenberger, M. L., Newton, M. A., Shelton, G. H., Ott, R. L., and

Guttorp, P. (1990). Evidence for the maintenance of hematopoiesis in a large animal by the sequential activation of stem-cell clones. Proc Natl Acad Sci U S A 87, 9062-

9066.

Acosta, S., Lavarino, C., Paris, R., Garcia, I., de Torres, C., Rodriguez, E., Beleta, H., and Mora, J. (2009). Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells. BMC Dev Biol

9, 12.

Akashi, K., Traver, D., Miyamoto, T., and Weissman, I. L. (2000). A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193-

197.

Al-Hajj, M., and Clarke, M. F. (2004). Self-renewal and solid tumor stem cells.

Oncogene 23, 7274-7282.

Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., and Clarke, M. F.

(2003). Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad

Sci U S A 100, 3983-3988.

Alvarez-Buylla, A., and Lois, C. (1995). Neuronal stem cells in the brain of adult vertebrates. Stem Cells 13, 263-272.

152

Ambros, I. M., and Ambros, P. F. (1995). Schwann cells in neuroblastoma. Eur J

Cancer 31A, 429-434.

Anderson, D. J. (1989). The neural crest cell lineage problem: neuropoiesis? Neuron 3,

1-12.

Anderson, D. J. (1993). Cell fate determination in the peripheral nervous system: the

sympathoadrenal progenitor. J Neurobiol 24, 185-198.

Antonchuk, J., Sauvageau, G., and Humphries, R. K. (2002). HOXB4-induced

expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39-45.

Aprelikova, O., Xiong, Y., and Liu, E. T. (1995). Both p16 and p21 families of

cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin- dependent kinases by the CDK-activating kinase. J Biol Chem 270, 18195-18197.

Asselin-Labat, M. L., Sutherland, K. D., Barker, H., Thomas, R., Shackleton, M.,

Forrest, N. C., Hartley, L., Robb, L., Grosveld, F. G., van der Wees, J., et al. (2007).

Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 9, 201-209.

Batlle, E., Henderson, J. T., Beghtel, H., van den Born, M. M., Sancho, E., Huls, G.,

Meeldijk, J., Robertson, J., van de Wetering, M., Pawson, T., and Clevers, H. (2002).

Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by

controlling the expression of EphB/ephrinB. Cell 111, 251-263.

Baudoin, C., Meneguzzi, G., Portier, M. M., Demarchez, M., Bernerd, F., Pisani, A.,

and Ortonne, J. P. (1993). Peripherin, a neuronal intermediate protein, is stably

153

expressed by neuroendocrine carcinomas of the skin, their xenograft on nude mice,

and the corresponding primary cultures. Cancer Res 53, 1175-1181.

Belinsky, S. A., Nikula, K. J., Palmisano, W. A., Michels, R., Saccomanno, G.,

Gabrielson, E., Baylin, S. B., and Herman, J. G. (1998). Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early

diagnosis. Proc Natl Acad Sci U S A 95, 11891-11896.

Birren, S. J., and Anderson, D. J. (1990). A v-myc-immortalized sympathoadrenal

progenitor cell line in which neuronal differentiation is initiated by FGF but not NGF.

Neuron 4, 189-201.

Bjerkvig, R., Tysnes, B. B., Aboody, K. S., Najbauer, J., and Terzis, A. J. (2005).

Opinion: the origin of the cancer stem cell: current controversies and new insights.

Nat Rev Cancer 5, 899-904.

Blair, A., Hogge, D. E., Ailles, L. E., Lansdorp, P. M., and Sutherland, H. J. (1997).

Lack of expression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term

proliferative ability in vitro and in vivo. Blood 89, 3104-3112.

Bonnet, D., and Dick, J. E. (1997). Human acute myeloid leukemia is organized as a

hierarchy that originates from a primitive hematopoietic cell. Nat Med 3, 730-737.

Britsch, S., Goerich, D. E., Riethmacher, D., Peirano, R. I., Rossner, M., Nave, K. A.,

Birchmeier, C., and Wegner, M. (2001). The transcription factor Sox10 is a key

regulator of peripheral glial development. Genes Dev 15, 66-78.

Brodeur, G. M. (2003). Neuroblastoma: biological insights into a clinical enigma. Nat

Rev Cancer 3, 203-216.

154

Canete, A., Gerrard, M., Rubie, H., Castel, V., Di Cataldo, A., Munzer, C., Ladenstein,

R., Brichard, B., Bermudez, J. D., Couturier, J., et al. (2009). Poor survival for infants with MYCN-amplified metastatic neuroblastoma despite intensified treatment: the

International Society of Paediatric Oncology European Neuroblastoma Experience. J

Clin Oncol 27, 1014-1019.

Carnahan, J. F., and Patterson, P. H. (1991). Isolation of the progenitor cells of the sympathoadrenal lineage from embryonic sympathetic ganglia with the SA monoclonal antibodies. J Neurosci 11, 3520-3530.

Cassady, J. R. (1984). A hypothesis to explain the enigmatic natural history of neuroblastoma. Med Pediatr Oncol 12, 64-67.

Castleberry, R. P. (1997). Neuroblastoma. Eur J Cancer 33, 1430-1437; discussion

1437-1438.

Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith,

A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643-655.

Chanas-Sacre, G., Rogister, B., Moonen, G., and Leprince, P. (2000). Radial glia phenotype: origin, regulation, and transdifferentiation. J Neurosci Res 61, 357-363.

Chapman, E. R., An, S., Barton, N., and Jahn, R. (1994). SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J

Biol Chem 269, 27427-27432.

Charron, F., and Nemer, M. (1999). GATA transcription factors and cardiac development. Semin Cell Dev Biol 10, 85-91.

155

Chau, B. N., and Wang, J. Y. (2003). Coordinated regulation of life and death by RB.

Nat Rev Cancer 3, 130-138.

Chaudhary, P. M., and Roninson, I. B. (1991). Expression and activity of P-

glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 66,

85-94.

Cheng, T., Rodrigues, N., Dombkowski, D., Stier, S., and Scadden, D. T. (2000a).

Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nat Med

6, 1235-1240.

Cheng, T., Rodrigues, N., Shen, H., Yang, Y., Dombkowski, D., Sykes, M., and

Scadden, D. T. (2000b). Hematopoietic stem cell quiescence maintained by

p21cip1/waf1. Science 287, 1804-1808.

Chong, J. L., Tsai, S. Y., Sharma, N., Opavsky, R., Price, R., Wu, L., Fernandez, S.

A., and Leone, G. (2009). E2f3a and E2f3b contribute to the control of cell proliferation and mouse development. Mol Cell Biol 29, 414-424.

Chung, E. J., Hwang, S. G., Nguyen, P., Lee, S., Kim, J. S., Kim, J. W., Henkart, P.

A., Bottaro, D. P., Soon, L., Bonvini, P., et al. (2002). Regulation of leukemic cell

adhesion, proliferation, and survival by beta-catenin. Blood 100, 982-990.

Cocchia, D., and Michetti, F. (1981). S-100 antigen in satellite cells of the adrenal

medulla and the superior cervical ganglion of the rat. An immunochemical and

immunocytochemical study. Cell Tissue Res 215, 103-112.

156

Collecchi, P., Santoni, T., Gnesi, E., Giuseppe Naccarato, A., Passoni, A., Rocchetta,

M., Danesi, R., and Bevilacqua, G. (2000). Cyclins of phases G1, S and G2/M are

overexpressed in aneuploid mammary carcinomas. Cytometry 42, 254-260.

Conner, E. A., Lemmer, E. R., Omori, M., Wirth, P. J., Factor, V. M., and

Thorgeirsson, S. S. (2000). Dual functions of E2F-1 in a transgenic mouse model of

liver carcinogenesis. Oncogene 19, 5054-5062.

Coughlin, M. D., Boyer, D. M., and Black, I. B. (1977). Embryologic development of

a mouse sympathetic ganglion in vivo and in vitro. Proc Natl Acad Sci U S A 74,

3438-3442.

Cozzio, A., Passegue, E., Ayton, P. M., Karsunky, H., Cleary, M. L., and Weissman, I.

L. (2003). Similar MLL-associated leukemias arising from self-renewing stem cells

and short-lived myeloid progenitors. Genes Dev 17, 3029-3035.

Crispino, J. D., Lodish, M. B., Thurberg, B. L., Litovsky, S. H., Collins, T.,

Molkentin, J. D., and Orkin, S. H. (2001). Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes

Dev 15, 839-844.

Cui, H., Hu, B., Li, T., Ma, J., Alam, G., Gunning, W. T., and Ding, H. F. (2007).

Bmi-1 is essential for the tumorigenicity of neuroblastoma cells. Am J Pathol 170,

1370-1378.

Cui, H., Ma, J., Ding, J., Li, T., Alam, G., and Ding, H. F. (2006). Bmi-1 regulates the

differentiation and clonogenic self-renewal of I-type neuroblastoma cells in a

concentration-dependent manner. J Biol Chem 281, 34696-34704.

157

Dahlstrand, J., Lardelli, M., and Lendahl, U. (1995). Nestin mRNA expression

correlates with the central nervous system progenitor cell state in many, but not all,

regions of developing central nervous system. Brain Res Dev Brain Res 84, 109-129.

Daser, A., and Rabbitts, T. H. (2004). Extending the repertoire of the mixed-lineage

leukemia gene MLL in leukemogenesis. Genes Dev 18, 965-974.

Davidoff, A. M., Humphrey, P. A., Iglehart, J. D., and Marks, J. R. (1991). Genetic

basis for p53 overexpression in human breast cancer. Proc Natl Acad Sci U S A 88,

5006-5010.

de Boer, C. J., van Krieken, J. H., Schuuring, E., and Kluin, P. M. (1997). Bcl-

1/cyclin D1 in malignant lymphoma. Ann Oncol 8 Suppl 2, 109-117.

Di Cristofano, A., and Pandolfi, P. P. (2000). The multiple roles of PTEN in tumor

suppression. Cell 100, 387-390.

Diehl, J. A. (2002). Cycling to cancer with cyclin D1. Cancer Biol Ther 1, 226-231.

Donovan, P. J. (1994). Growth factor regulation of mouse primordial germ cell

development. Curr Top Dev Biol 29, 189-225.

Dontu, G. (2008). Breast cancer stem cell markers - the rocky road to clinical

applications. Breast Cancer Res 10, 110.

Dottori, M., Gross, M. K., Labosky, P., and Goulding, M. (2001). The winged-helix

transcription factor Foxd3 suppresses interneuron differentiation and promotes neural

crest cell fate. Development 128, 4127-4138.

158

Drobnjak, M., Osman, I., Scher, H. I., Fazzari, M., and Cordon-Cardo, C. (2000).

Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone.

Clin Cancer Res 6, 1891-1895.

Durbec, P. L., Larsson-Blomberg, L. B., Schuchardt, A., Costantini, F., and Pachnis,

V. (1996). Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. Development 122, 349-358.

Durocher, D., Charron, F., Warren, R., Schwartz, R. J., and Nemer, M. (1997). The

cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J 16,

5687-5696.

Ermini, F. V., Grathwohl, S., Radde, R., Yamaguchi, M., Staufenbiel, M., Palmer, T.

D., and Jucker, M. (2008). Neurogenesis and alterations of neural stem cells in mouse

models of cerebral amyloidosis. Am J Pathol 172, 1520-1528.

Evangelopoulos, M. E., Weis, J., and Kruttgen, A. (2004). Neurotrophin effects on

neuroblastoma cells: correlation with trk and p75NTR expression and influence of

Trk receptor bodies. J Neurooncol 66, 101-110.

Feng, L., Hatten, M. E., and Heintz, N. (1994). Brain lipid-binding protein (BLBP): a

novel signaling system in the developing mammalian CNS. Neuron 12, 895-908.

Fillmore, C., and Kuperwasser, C. (2007). Human breast cancer stem cell markers

CD44 and CD24: enriching for cells with functional properties in mice or in man?

Breast Cancer Res 9, 303.

159

Flora, A., Lucchetti, H., Benfante, R., Goridis, C., Clementi, F., and Fornasari, D.

(2001). Sp proteins and Phox2b regulate the expression of the human Phox2a gene. J

Neurosci 21, 7037-7045.

Fox, A. H., Liew, C., Holmes, M., Kowalski, K., Mackay, J., and Crossley, M. (1999).

Transcriptional cofactors of the FOG family interact with GATA proteins by means of

multiple zinc fingers. EMBO J 18, 2812-2822.

Francis, N. J., and Landis, S. C. (1999). Cellular and molecular determinants of

sympathetic neuron development. Annu Rev Neurosci 22, 541-566.

Freedberg, D. E., Rigas, S. H., Russak, J., Gai, W., Kaplow, M., Osman, I., Turner, F.,

Randerson-Moor, J. A., Houghton, A., Busam, K., et al. (2008). Frequent p16-

independent inactivation of p14ARF in human melanoma. J Natl Cancer Inst 100,

784-795.

Gage, F. H., Ray, J., and Fisher, L. J. (1995). Isolation, characterization, and use of

stem cells from the CNS. Annu Rev Neurosci 18, 159-192.

Galderisi, U., Jori, F. P., and Giordano, A. (2003). Cell cycle regulation and neural

differentiation. Oncogene 22, 5208-5219.

Galizia, G., Ferraraccio, F., Lieto, E., Orditura, M., Castellano, P., Imperatore, V.,

Romano, C., Vollaro, M., Agostini, B., Pignatelli, C., and De Vita, F. (2004).

Prognostic value of p27, p53, and vascular endothelial growth factor in Dukes A and

B colon cancer patients undergoing potentially curative surgery. Dis Colon Rectum 47,

1904-1914.

160

Garg, V., Kathiriya, I. S., Barnes, R., Schluterman, M. K., King, I. N., Butler, C. A.,

Rothrock, C. R., Eapen, R. S., Hirayama-Yamada, K., Joo, K., et al. (2003). GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5.

Nature 424, 443-447.

Gat, U., DasGupta, R., Degenstein, L., and Fuchs, E. (1998). De Novo hair follicle

morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.

Cell 95, 605-614.

George, K. M., Leonard, M. W., Roth, M. E., Lieuw, K. H., Kioussis, D., Grosveld, F.,

and Engel, J. D. (1994). Embryonic expression and cloning of the murine GATA-3

gene. Development 120, 2673-2686.

Glebova, N. O., and Ginty, D. D. (2005). Growth and survival signals controlling

sympathetic nervous system development. Annu Rev Neurosci 28, 191-222.

Godlewski, J., Nowicki, M. O., Bronisz, A., Williams, S., Otsuki, A., Nuovo, G.,

Raychaudhury, A., Newton, H. B., Chiocca, E. A., and Lawler, S. (2008). Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res 68, 9125-9130.

Gordon, D. F., Lewis, S. R., Haugen, B. R., James, R. A., McDermott, M. T., Wood,

W. M., and Ridgway, E. C. (1997). Pit-1 and GATA-2 interact and functionally

cooperate to activate the thyrotropin beta-subunit promoter. J Biol Chem 272, 24339-

24347.

Goridis, C., and Rohrer, H. (2002). Specification of catecholaminergic and

serotonergic neurons. Nat Rev Neurosci 3, 531-541.

161

Gross, C. G. (2000). Neurogenesis in the adult brain: death of a dogma. Nat Rev

Neurosci 1, 67-73.

Groszer, M., Erickson, R., Scripture-Adams, D. D., Dougherty, J. D., Le Belle, J.,

Zack, J. A., Geschwind, D. H., Liu, X., Kornblum, H. I., and Wu, H. (2006). PTEN negatively regulates neural stem cell self-renewal by modulating G0-G1 cell cycle entry. Proc Natl Acad Sci U S A 103, 111-116.

Grote, D., Boualia, S. K., Souabni, A., Merkel, C., Chi, X., Costantini, F., Carroll, T.,

and Bouchard, M. (2008). Gata3 acts downstream of beta-catenin signaling to prevent

ectopic metanephric kidney induction. PLoS Genet 4, e1000316.

Guillemot, F., and Joyner, A. L. (1993). Dynamic expression of the murine Achaete-

Scute homologue Mash-1 in the developing nervous system. Mech Dev 42, 171-185.

Guillemot, F., Lo, L. C., Johnson, J. E., Auerbach, A., Anderson, D. J., and Joyner, A.

L. (1993). Mammalian achaete-scute homolog 1 is required for the early development

of olfactory and autonomic neurons. Cell 75, 463-476.

Gulbinas, A., Berberat, P. O., Dambrauskas, Z., Giese, T., Giese, N., Autschbach, F.,

Kleeff, J., Meuer, S., Buchler, M. W., and Friess, H. (2006). Aberrant gata-3

expression in human pancreatic cancer. J Histochem Cytochem 54, 161-169.

Gunawardena, R. W., Fox, S. R., Siddiqui, H., and Knudsen, E. S. (2007). SWI/SNF

activity is required for the repression of deoxyribonucleotide triphosphate metabolic

enzymes via the recruitment of mSin3B. J Biol Chem 282, 20116-20123.

Hagen, E. C., Vennegoor, C., Schlingemann, R. O., van der Velde, E. A., and Ruiter,

D. J. (1986). Correlation of histopathological characteristics with staining patterns in

162

human melanoma assessed by (monoclonal) antibodies reactive on paraffin sections.

Histopathology 10, 689-700.

Hall, A. K., and Landis, S. C. (1991). Early commitment of precursor cells from the rat superior cervical ganglion to neuronal or nonneuronal fates. Neuron 6, 741-752.

Hall, A. K., and Landis, S. C. (1992). Division and migration of satellite glia in the embryonic rat superior cervical ganglion. J Neurocytol 21, 635-647.

Hall, P. A., and Watt, F. M. (1989). Stem cells: the generation and maintenance of cellular diversity. Development 106, 619-633.

Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.

Hanna, L. A., Foreman, R. K., Tarasenko, I. A., Kessler, D. S., and Labosky, P. A.

(2002). Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Dev 16, 2650-2661.

He, G., Siddik, Z. H., Huang, Z., Wang, R., Koomen, J., Kobayashi, R., Khokhar, A.

R., and Kuang, J. (2005). Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities. Oncogene 24, 2929-2943.

Hendry, I. A. (1977). Cell division in the developing sympathetic nervous system. J

Neurocytol 6, 299-309.

Hirschmann-Jax, C., Foster, A. E., Wulf, G. G., Nuchtern, J. G., Jax, T. W., Gobel, U.,

Goodell, M. A., and Brenner, M. K. (2004). A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A 101,

14228-14233.

163

Hirst, G. D., and McLachlan, E. M. (1984). Post-natal development of ganglia in the

lower lumbar sympathetic chain of the rat. J Physiol 349, 119-134.

Hitoshi, S., Alexson, T., Tropepe, V., Donoviel, D., Elia, A. J., Nye, J. S., Conlon, R.

A., Mak, T. W., Bernstein, A., and van der Kooy, D. (2002). Notch pathway

molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 16, 846-858.

Hoch, R. V., Thompson, D. A., Baker, R. J., and Weigel, R. J. (1999). GATA-3 is

expressed in association with estrogen receptor in breast cancer. Int J Cancer 84, 122-

128.

Hockfield, S., and McKay, R. D. (1985). Identification of major cell classes in the

developing mammalian nervous system. J Neurosci 5, 3310-3328.

Hogarty, M. D. (2003). The requirement for evasion of programmed cell death in

neuroblastomas with MYCN amplification. Cancer Lett 197, 173-179.

Holmes, M., Turner, J., Fox, A., Chisholm, O., Crossley, M., and Chong, B. (1999).

hFOG-2, a novel zinc finger protein, binds the co-repressor mCtBP2 and modulates

GATA-mediated activation. J Biol Chem 274, 23491-23498.

Hong, S. J., Choi, H. J., Hong, S., Huh, Y., Chae, H., and Kim, K. S. (2008).

Transcription factor GATA-3 regulates the transcriptional activity of dopamine beta-

hydroxylase by interacting with Sp1 and AP4. Neurochem Res 33, 1821-1831.

Hong, S. J., Huh, Y., Chae, H., Hong, S., Lardaro, T., and Kim, K. S. (2006). GATA-

3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with

CREB. J Neurochem 98, 773-781.

164

Howard, M. J. (2005). Mechanisms and perspectives on differentiation of autonomic

neurons. Dev Biol 277, 271-286.

Howard, M. J., Stanke, M., Schneider, C., Wu, X., and Rohrer, H. (2000). The

transcription factor dHAND is a downstream effector of BMPs in sympathetic neuron

specification. Development 127, 4073-4081.

Hsi, E. D., Zukerberg, L. R., Yang, W. I., and Arnold, A. (1996). Cyclin D1/PRAD1

expression in parathyroid adenomas: an immunohistochemical study. J Clin

Endocrinol Metab 81, 1736-1739.

Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G., and Birchmeier, W. (2001).

beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the

skin. Cell 105, 533-545.

Huttenbach, Y., Prieto, V. G., and Reed, J. A. (2002). Desmoplastic and spindle cell

melanomas express protein markers of the neural crest but not of later committed

stages of Schwann cell differentiation. J Cutan Pathol 29, 562-568.

Hwang, E. S., Szabo, S. J., Schwartzberg, P. L., and Glimcher, L. H. (2005). T helper

cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science 307,

430-433.

Imura, T., Kornblum, H. I., and Sofroniew, M. V. (2003). The predominant neural

stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain

expresses GFAP. J Neurosci 23, 2824-2832.

165

Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999a).

The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and

senescence through the ink4a locus. Nature 397, 164-168.

Jacobs, J. J., Scheijen, B., Voncken, J. W., Kieboom, K., Berns, A., and van Lohuizen,

M. (1999b). Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-

induced apoptosis via INK4a/ARF. Genes Dev 13, 2678-2690.

Jacobs, J. J., and van Lohuizen, M. (2002). Polycomb repression: from cellular

memory to cellular proliferation and cancer. Biochim Biophys Acta 1602, 151-161.

Jacquemier, J., Ginestier, C., Rougemont, J., Bardou, V. J., Charafe-Jauffret, E.,

Geneix, J., Adelaide, J., Koki, A., Houvenaeghel, G., Hassoun, J., et al. (2005).

Protein expression profiling identifies subclasses of breast cancer and predicts

prognosis. Cancer Res 65, 767-779.

Jenssen, T. K., Kuo, W. P., Stokke, T., and Hovig, E. (2002). Associations between

gene expressions in breast cancer and patient survival. Hum Genet 111, 411-420.

Jessen, K. R., and Mirsky, R. (2005). The origin and development of glial cells in

peripheral nerves. Nat Rev Neurosci 6, 671-682.

Jimenez, P., Saner, K., Mayhew, B., and Rainey, W. E. (2003). GATA-6 is expressed

in the human adrenal and regulates transcription of genes required for adrenal

androgen biosynthesis. Endocrinology 144, 4285-4288.

Johnstone, R. W., Cretney, E., and Smyth, M. J. (1999). P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent, cell death.

Blood 93, 1075-1085.

166

Jordan, C. T., Upchurch, D., Szilvassy, S. J., Guzman, M. L., Howard, D. S.,

Pettigrew, A. L., Meyerrose, T., Rossi, R., Grimes, B., Rizzieri, D. A., et al. (2000).

The interleukin-3 receptor alpha chain is a unique marker for human acute

myelogenous leukemia stem cells. Leukemia 14, 1777-1784.

Karanu, F. N., Murdoch, B., Gallacher, L., Wu, D. M., Koremoto, M., Sakano, S., and

Bhatia, M. (2000). The notch ligand jagged-1 represents a novel growth factor of

human hematopoietic stem cells. J Exp Med 192, 1365-1372.

Kaufman, C. K., Zhou, P., Pasolli, H. A., Rendl, M., Bolotin, D., Lim, K. C., Dai, X.,

Alegre, M. L., and Fuchs, E. (2003). GATA-3: an unexpected regulator of cell lineage determination in skin. Genes Dev 17, 2108-2122.

Kenney, A. M., and Rowitch, D. H. (2000). Sonic hedgehog promotes G(1) cyclin

expression and sustained cell cycle progression in mammalian neuronal precursors.

Mol Cell Biol 20, 9055-9067.

Key, G., Becker, M. H., Baron, B., Duchrow, M., Schluter, C., Flad, H. D., and

Gerdes, J. (1993). New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3)

generated against bacterially expressed parts of the Ki-67 cDNA containing three 62

base pair repetitive elements encoding for the Ki-67 epitope. Lab Invest 68, 629-636.

Kim, J., Lo, L., Dormand, E., and Anderson, D. J. (2003). SOX10 maintains

multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron

38, 17-31.

Kinzler, K. W., and Vogelstein, B. (1996). Lessons from hereditary colorectal cancer.

Cell 87, 159-170.

167

Kirby, M. L., and Gilmore, S. A. (1976). A correlative histofluorescence and light microscopic study of the formation of the sympathetic trunks in chick embryos. Anat

Rec 186, 437-449.

Klopfleisch, R., and Gruber, A. D. (2009). Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands. Res Vet Sci.

Knudsen, K. E., Diehl, J. A., Haiman, C. A., and Knudsen, E. S. (2006). Cyclin D1: polymorphism, aberrant splicing and cancer risk. Oncogene 25, 1620-1628.

Ko, L. J., and Engel, J. D. (1993). DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol 13, 4011-4022.

Koljonen, V., Tukiainen, E., Haglund, C., and Bohling, T. (2006). Cell cycle control by p21, p27 and p53 in Merkel cell carcinoma. Anticancer Res 26, 2209-2212.

Kos, R., Reedy, M. V., Johnson, R. L., and Erickson, C. A. (2001). The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development 128, 1467-1479.

Kouros-Mehr, H., Bechis, S. K., Slorach, E. M., Littlepage, L. E., Egeblad, M., Ewald,

A. J., Pai, S. Y., Ho, I. C., and Werb, Z. (2008). GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. Cancer Cell 13, 141-152.

Kouros-Mehr, H., Slorach, E. M., Sternlicht, M. D., and Werb, Z. (2006). GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell 127,

1041-1055.

168

Kristiansen, G., Sammar, M., and Altevogt, P. (2004). Tumour biological aspects of

CD24, a mucin-like adhesion molecule. J Mol Histol 35, 255-262.

Krivtsov, A. V., Twomey, D., Feng, Z., Stubbs, M. C., Wang, Y., Faber, J., Levine, J.

E., Wang, J., Hahn, W. C., Gilliland, D. G., et al. (2006). Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818-

822.

Kurek, D., Garinis, G. A., van Doorninck, J. H., van der Wees, J., and Grosveld, F. G.

(2007). Transcriptome and phenotypic analysis reveals Gata3-dependent signalling pathways in murine hair follicles. Development 134, 261-272.

Kurtz, A., Zimmer, A., Schnutgen, F., Bruning, G., Spener, F., and Muller, T. (1994).

The expression pattern of a novel gene encoding brain-fatty acid binding protein correlates with neuronal and glial cell development. Development 120, 2637-2649.

Labastie, M. C., Catala, M., Gregoire, J. M., and Peault, B. (1995). The GATA-3 gene is expressed during human kidney embryogenesis. Kidney Int 47, 1597-1603.

LaBonne, C., and Bronner-Fraser, M. (1998). Induction and patterning of the neural crest, a stem cell-like precursor population. J Neurobiol 36, 175-189.

Lai, K., Kaspar, B. K., Gage, F. H., and Schaffer, D. V. (2003). Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat Neurosci 6, 21-

27.

169

Laitinen, M. P., Anttonen, M., Ketola, I., Wilson, D. B., Ritvos, O., Butzow, R., and

Heikinheimo, M. (2000). Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors. J Clin Endocrinol Metab 85, 3476-3483.

Lapidot, T., Sirard, C., Vormoor, J., Murdoch, B., Hoang, T., Caceres-Cortes, J.,

Minden, M., Paterson, B., Caligiuri, M. A., and Dick, J. E. (1994). A cell initiating

human acute myeloid leukaemia after transplantation into SCID mice. Nature 367,

645-648.

Lavker, R. M., Miller, S., Wilson, C., Cotsarelis, G., Wei, Z. G., Yang, J. S., and Sun,

T. T. (1993). Hair follicle stem cells: their location, role in hair cycle, and

involvement in skin tumor formation. J Invest Dermatol 101, 16S-26S.

Le Douarin, N. M., and Dupin, E. (1993). Cell lineage analysis in neural crest

ontogeny. J Neurobiol 24, 146-161.

Le Douarin, N. M., and Kalcheim, C. (1999). The neural crest, Vol 36, 2nd edn

(Cambridge, Cambridge University Press).

Lendahl, U., Zimmerman, L. B., and McKay, R. D. (1990). CNS stem cells express a new class of intermediate filament protein. Cell 60, 585-595.

Lessard, J., and Sauvageau, G. (2003). Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423, 255-260.

Leung, C., Lingbeek, M., Shakhova, O., Liu, J., Tanger, E., Saremaslani, P., Van

Lohuizen, M., and Marino, S. (2004). Bmi1 is essential for cerebellar development

and is overexpressed in human medulloblastomas. Nature 428, 337-341.

170

Li, C., Heidt, D. G., Dalerba, P., Burant, C. F., Zhang, L., Adsay, V., Wicha, M.,

Clarke, M. F., and Simeone, D. M. (2007). Identification of pancreatic cancer stem cells. Cancer Res 67, 1030-1037.

Li, J., Simpson, L., Takahashi, M., Miliaresis, C., Myers, M. P., Tonks, N., and

Parsons, R. (1998). The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res 58, 5667-5672.

Lillevali, K., Haugas, M., Matilainen, T., Pussinen, C., Karis, A., and Salminen, M.

(2006). Gata3 is required for early morphogenesis and Fgf10 expression during otic development. Mech Dev 123, 415-429.

Lillevali, K., Matilainen, T., Karis, A., and Salminen, M. (2004). Partially overlapping expression of Gata2 and Gata3 during inner ear development. Dev Dyn

231, 775-781.

Lim, K. C., Lakshmanan, G., Crawford, S. E., Gu, Y., Grosveld, F., and Engel, J. D.

(2000). Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nat Genet 25, 209-212.

Lo, L., Tiveron, M. C., and Anderson, D. J. (1998). MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity. Development 125, 609-

620.

Lotayef, M., Wilson, G. D., Daley, F. M., Awwad, H. K., and Shouman, T. (2000).

Aberrant expression of cyclin A in head and neck cancer. Br J Cancer 83, 30-34.

171

Lothian, C., and Lendahl, U. (1997). An evolutionarily conserved region in the second

intron of the human nestin gene directs gene expression to CNS progenitor cells and

to early neural crest cells. Eur J Neurosci 9, 452-462.

Lowry, J. A., and Atchley, W. R. (2000). Molecular evolution of the GATA family of

transcription factors: conservation within the DNA-binding domain. J Mol Evol 50,

103-115.

Lu, J. R., McKinsey, T. A., Xu, H., Wang, D. Z., Richardson, J. A., and Olson, E. N.

(1999). FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors.

Mol Cell Biol 19, 4495-4502.

Lutz, W., Stohr, M., Schurmann, J., Wenzel, A., Lohr, A., and Schwab, M. (1996).

Conditional expression of N-myc in human neuroblastoma cells increases expression

of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-

phase early after mitogenic stimulation of quiescent cells. Oncogene 13, 803-812.

Ma, G. T., Roth, M. E., Groskopf, J. C., Tsai, F. Y., Orkin, S. H., Grosveld, F., Engel,

J. D., and Linzer, D. I. (1997). GATA-2 and GATA-3 regulate trophoblast-specific

gene expression in vivo. Development 124, 907-914.

Machold, R., Hayashi, S., Rutlin, M., Muzumdar, M. D., Nery, S., Corbin, J. G.,

Gritli-Linde, A., Dellovade, T., Porter, J. A., Rubin, L. L., et al. (2003). Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches.

Neuron 39, 937-950.

Maitland, N. J., and Collins, A. T. (2008). Prostate cancer stem cells: a new target for

therapy. J Clin Oncol 26, 2862-2870.

172

Margolis, J., and Spradling, A. (1995). Identification and behavior of epithelial stem

cells in the Drosophila ovary. Development 121, 3797-3807.

Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J., and Berns, A. (2000).

Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev 14, 994-1004.

Martin, D. I., and Orkin, S. H. (1990). Transcriptional activation and DNA binding by

the erythroid factor GF-1/NF-E1/Eryf 1. Genes Dev 4, 1886-1898.

Matsuoka, K., Nomura, K., and Hoshino, T. (1990). Mutagenic effects of brief

exposure to bromodeoxyuridine on mouse FM3A cells. Cell Tissue Kinet 23, 495-503.

McMahon, H. T., and Sudhof, T. C. (1995). Synaptic core complex of synaptobrevin,

syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem 270,

2213-2217.

Mehra, R., Varambally, S., Ding, L., Shen, R., Sabel, M. S., Ghosh, D., Chinnaiyan,

A. M., and Kleer, C. G. (2005). Identification of GATA3 as a breast cancer prognostic

marker by global gene expression meta-analysis. Cancer Res 65, 11259-11264.

Merika, M., and Orkin, S. H. (1993). DNA-binding specificity of GATA family

transcription factors. Mol Cell Biol 13, 3999-4010.

Merika, M., and Orkin, S. H. (1995). Functional synergy and physical interactions of

the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and

EKLF. Mol Cell Biol 15, 2437-2447.

173

Milde-Langosch, K., Bamberger, A. M., Rieck, G., Kelp, B., and Loning, T. (2001).

Overexpression of the p16 cell cycle inhibitor in breast cancer is associated with a

more malignant phenotype. Breast Cancer Res Treat 67, 61-70.

Minegishi, N., Morita, S., Minegishi, M., Tsuchiya, S., Konno, T., Hayashi, N., and

Yamamoto, M. (1997). Expression of GATA transcription factors in myelogenous

and lymphoblastic leukemia cells. Int J Hematol 65, 239-249.

Ming, G. L., and Song, H. (2005). Adult neurogenesis in the mammalian central

nervous system. Annu Rev Neurosci 28, 223-250.

Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K.,

Maruyama, M., Maeda, M., and Yamanaka, S. (2003). The homeoprotein Nanog is

required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113,

631-642.

Miyazawa, K., Himi, T., Garcia, V., Yamagishi, H., Sato, S., and Ishizaki, Y. (2000).

A role for p27/Kip1 in the control of cerebellar granule cell precursor proliferation. J

Neurosci 20, 5756-5763.

Molenaar, J. J., van Sluis, P., Boon, K., Versteeg, R., and Caron, H. N. (2003).

Rearrangements and increased expression of cyclin D1 (CCND1) in neuroblastoma.

Genes Chromosomes Cancer 36, 242-249.

Molkentin, J. D. (2000). The zinc finger-containing transcription factors GATA-4, -5,

and -6. Ubiquitously expressed regulators of tissue-specific gene expression. J Biol

Chem 275, 38949-38952.

174

Molofsky, A. V., He, S., Bydon, M., Morrison, S. J., and Pardal, R. (2005). Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev

19, 1432-1437.

Molofsky, A. V., Pardal, R., Iwashita, T., Park, I. K., Clarke, M. F., and Morrison, S.

J. (2003). Bmi-1 dependence distinguishes neural stem cell self-renewal from

progenitor proliferation. Nature 425, 962-967.

Mora, J., Cheung, N. K., Juan, G., Illei, P., Cheung, I., Akram, M., Chi, S., Ladanyi,

M., Cordon-Cardo, C., and Gerald, W. L. (2001). Neuroblastic and Schwannian

stromal cells of neuroblastoma are derived from a tumoral progenitor cell. Cancer Res

61, 6892-6898.

Moriguchi, T., Takako, N., Hamada, M., Maeda, A., Fujioka, Y., Kuroha, T., Huber,

R. E., Hasegawa, S. L., Rao, A., Yamamoto, M., et al. (2006). Gata3 participates in a

complex transcriptional feedback network to regulate sympathoadrenal differentiation.

Development 133, 3871-3881.

Morikawa, Y., Dai, Y. S., Hao, J., Bonin, C., Hwang, S., and Cserjesi, P. (2005). The

basic helix-loop-helix factor Hand 2 regulates autonomic nervous system development. Dev Dyn 234, 613-621.

Morin, S., Charron, F., Robitaille, L., and Nemer, M. (2000). GATA-dependent

recruitment of MEF2 proteins to target promoters. EMBO J 19, 2046-2055.

Morrison, S. J., Hemmati, H. D., Wandycz, A. M., and Weissman, I. L. (1995a). The

purification and characterization of fetal liver hematopoietic stem cells. Proc Natl

Acad Sci U S A 92, 10302-10306. 175

Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C., Weinmaster, G., and

Anderson, D. J. (2000). Transient Notch activation initiates an irreversible switch

from neurogenesis to gliogenesis by neural crest stem cells. Cell 101, 499-510.

Morrison, S. J., Uchida, N., and Weissman, I. L. (1995b). The biology of

hematopoietic stem cells. Annu Rev Cell Dev Biol 11, 35-71.

Morrison, S. J., Wandycz, A. M., Akashi, K., Globerson, A., and Weissman, I. L.

(1996). The aging of hematopoietic stem cells. Nat Med 2, 1011-1016.

Morrison, S. J., and Weissman, I. L. (1994). The long-term repopulating subset of

hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1,

661-673.

Moser, A. R., Dove, W. F., Roth, K. A., and Gordon, J. I. (1992). The Min (multiple

intestinal neoplasia) mutation: its effect on gut epithelial cell differentiation and

interaction with a modifier system. J Cell Biol 116, 1517-1526.

Nakagawara, A. (1998). Molecular basis of spontaneous regression of neuroblastoma:

role of neurotrophic signals and genetic abnormalities. Hum Cell 11, 115-124.

Nardelli, J., Thiesson, D., Fujiwara, Y., Tsai, F. Y., and Orkin, S. H. (1999).

Expression and genetic interaction of transcription factors GATA-2 and GATA-3

during development of the mouse central nervous system. Dev Biol 210, 305-321.

176

Nawijn, M. C., Ferreira, R., Dingjan, G. M., Kahre, O., Drabek, D., Karis, A.,

Grosveld, F., and Hendriks, R. W. (2001). Enforced expression of GATA-3 during T

cell development inhibits maturation of CD8 single-positive cells and induces thymic

lymphoma in transgenic mice. J Immunol 167, 715-723.

Nichols, K. E., Crispino, J. D., Poncz, M., White, J. G., Orkin, S. H., Maris, J. M., and

Weiss, M. J. (2000). Familial dyserythropoietic anaemia and thrombocytopenia due to

an inherited mutation in GATA1. Nat Genet 24, 266-270.

Niwa, H., Burdon, T., Chambers, I., and Smith, A. (1998). Self-renewal of pluripotent

embryonic stem cells is mediated via activation of STAT3. Genes Dev 12, 2048-2060.

Novick, P., and Zerial, M. (1997). The diversity of Rab proteins in vesicle transport.

Curr Opin Cell Biol 9, 496-504.

Nozaki, M., Tada, M., Kobayashi, H., Zhang, C. L., Sawamura, Y., Abe, H., Ishii, N.,

and Van Meir, E. G. (1999). Roles of the functional loss of p53 and other genes in

astrocytoma tumorigenesis and progression. Neuro Oncol 1, 124-137.

O'Brien, C. A., Pollett, A., Gallinger, S., and Dick, J. E. (2007). A human colon

cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445,

106-110.

Ohta, H., Sawada, A., Kim, J. Y., Tokimasa, S., Nishiguchi, S., Humphries, R. K.,

Hara, J., and Takihara, Y. (2002). Polycomb group gene rae28 is required for

sustaining activity of hematopoietic stem cells. J Exp Med 195, 759-770.

Omichinski, J. G., Trainor, C., Evans, T., Gronenborn, A. M., Clore, G. M., and

Felsenfeld, G. (1993). A small single-"finger" peptide from the erythroid transcription

177

factor GATA-1 binds specifically to DNA as a zinc or iron complex. Proc Natl Acad

Sci U S A 90, 1676-1680.

Ono, Y., Fukuhara, N., and Yoshie, O. (1998). TAL1 and LIM-only proteins

synergistically induce retinaldehyde dehydrogenase 2 expression in T-cell acute

lymphoblastic leukemia by acting as cofactors for GATA3. Mol Cell Biol 18, 6939-

6950.

Pai, S. Y., Truitt, M. L., and Ho, I. C. (2004). GATA-3 deficiency abrogates the

development and maintenance of T helper type 2 cells. Proc Natl Acad Sci U S A 101,

1993-1998.

Pallis, M., and Russell, N. (2000). P-glycoprotein plays a drug-efflux-independent

role in augmenting cell survival in acute myeloblastic leukemia and is associated with

modulation of a sphingomyelin-ceramide apoptotic pathway. Blood 95, 2897-2904.

Palmieri, S. L., Peter, W., Hess, H., and Scholer, H. R. (1994). Oct-4 transcription

factor is differentially expressed in the mouse embryo during establishment of the first

two extraembryonic cell lineages involved in implantation. Dev Biol 166, 259-267.

Pandolfi, P. P., Roth, M. E., Karis, A., Leonard, M. W., Dzierzak, E., Grosveld, F. G.,

Engel, J. D., and Lindenbaum, M. H. (1995). Targeted disruption of the GATA3 gene

causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

Nat Genet 11, 40-44.

Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., Morrison,

S. J., and Clarke, M. F. (2003). Bmi-1 is required for maintenance of adult self-

renewing haematopoietic stem cells. Nature 423, 302-305.

178

Patient, R. K., and McGhee, J. D. (2002). The GATA family (vertebrates and

invertebrates). Curr Opin Genet Dev 12, 416-422.

Pattyn, A., Morin, X., Cremer, H., Goridis, C., and Brunet, J. F. (1997). Expression

and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 124, 4065-4075.

Pattyn, A., Morin, X., Cremer, H., Goridis, C., and Brunet, J. F. (1999). The

homeobox gene Phox2b is essential for the development of autonomic neural crest

derivatives. Nature 399, 366-370.

Pear, W. S., Aster, J. C., Scott, M. L., Hasserjian, R. P., Soffer, B., Sklar, J., and

Baltimore, D. (1996). Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J Exp Med 183,

2283-2291.

Pierce, A. M., Gimenez-Conti, I. B., Schneider-Broussard, R., Martinez, L. A., Conti,

C. J., and Johnson, D. G. (1998). Increased E2F1 activity induces skin tumors in mice heterozygous and nullizygous for p53. Proc Natl Acad Sci U S A 95, 8858-8863.

Pierce, A. M., Schneider-Broussard, R., Gimenez-Conti, I. B., Russell, J. L., Conti, C.

J., and Johnson, D. G. (1999). E2F1 has both oncogenic and tumor-suppressive

properties in a transgenic model. Mol Cell Biol 19, 6408-6414.

Pietsch, T., Waha, A., Koch, A., Kraus, J., Albrecht, S., Tonn, J., Sorensen, N.,

Berthold, F., Henk, B., Schmandt, N., et al. (1997). Medulloblastomas of the

desmoplastic variant carry mutations of the human homologue of Drosophila patched.

Cancer Res 57, 2085-2088.

179

Plaza-Menacho, I., Burzynski, G. M., de Groot, J. W., Eggen, B. J., and Hofstra, R. M.

(2006). Current concepts in RET-related genetics, signaling and therapeutics. Trends

Genet 22, 627-636.

Plutner, H., Cox, A. D., Pind, S., Khosravi-Far, R., Bourne, J. R., Schwaninger, R.,

Der, C. J., and Balch, W. E. (1991). Rab1b regulates vesicular transport between the

endoplasmic reticulum and successive Golgi compartments. J Cell Biol 115, 31-43.

Polakis, P. (1999). The oncogenic activation of beta-catenin. Curr Opin Genet Dev 9,

15-21.

Poncet, C., Frances, V., Gristina, R., Scheiner, C., Pellissier, J. F., and Figarella-

Branger, D. (1996). CD24, a glycosylphosphatidylinositol-anchored molecules is

transiently expressed during the development of human central nervous system and is

a marker of human neural cell lineage tumors. Acta Neuropathol 91, 400-408.

Portier, M. M., de Nechaud, B., and Gros, F. (1983). Peripherin, a new member of the

intermediate filament protein family. Dev Neurosci 6, 335-344.

Potten, C. S., and Loeffler, M. (1990). Stem cells: attributes, cycles, spirals, pitfalls

and uncertainties. Lessons for and from the crypt. Development 110, 1001-1020.

Powell, S. M., Zilz, N., Beazer-Barclay, Y., Bryan, T. M., Hamilton, S. R., Thibodeau,

S. N., Vogelstein, B., and Kinzler, K. W. (1992). APC mutations occur early during

colorectal tumorigenesis. Nature 359, 235-237.

Preston, S. L., Alison, M. R., Forbes, S. J., Direkze, N. C., Poulsom, R., and Wright,

N. A. (2003). The new stem cell biology: something for everyone. Mol Pathol 56, 86-

96.

180

Qiang, Y. W., Endo, Y., Rubin, J. S., and Rudikoff, S. (2003). Wnt signaling in B-cell neoplasia. Oncogene 22, 1536-1545.

Reissmann, E., Ernsberger, U., Francis-West, P. H., Rueger, D., Brickell, P. M., and

Rohrer, H. (1996). Involvement of bone morphogenetic protein-4 and bone

morphogenetic protein-7 in the differentiation of the adrenergic phenotype in

developing sympathetic neurons. Development 122, 2079-2088.

Reiter, R. E., Gu, Z., Watabe, T., Thomas, G., Szigeti, K., Davis, E., Wahl, M.,

Nisitani, S., Yamashiro, J., Le Beau, M. M., et al. (1998). Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer. Proc Natl Acad Sci U S A 95,

1735-1740.

Reynolds, C. P. (2004). Detection and treatment of minimal residual disease in high-

risk neuroblastoma. Pediatr Transplant 8 Suppl 5, 56-66.

Robert, N. M., Tremblay, J. J., and Viger, R. S. (2002). Friend of GATA (FOG)-1 and

FOG-2 differentially repress the GATA-dependent activity of multiple gonadal

promoters. Endocrinology 143, 3963-3973.

Ross, R. A., Biedler, J. L., and Spengler, B. A. (2003). A role for distinct cell types in

determining malignancy in human neuroblastoma cell lines and tumors. Cancer Lett

197, 35-39.

Ross, R. A., Hein, A. M., Braca, J. A., 3rd, Spengler, B. A., Biedler, J. L., and

Scammell, J. G. (2002). Glucocorticoids induce neuroendocrine cell differentiation and increase expression of N-myc in N-type human neuroblastoma cells. Oncol Res

13, 87-94.

181

Ross, R. A., and Spengler, B. A. (2007). Human neuroblastoma stem cells. Semin

Cancer Biol 17, 241-247.

Rychlik, J. L., Gerbasi, V., and Lewis, E. J. (2003). The interaction between dHAND and Arix at the dopamine beta-hydroxylase promoter region is independent of direct dHAND binding to DNA. J Biol Chem 278, 49652-49660.

Sancho-Tello, M., Valles, S., Montoliu, C., Renau-Piqueras, J., and Guerri, C. (1995).

Developmental pattern of GFAP and vimentin gene expression in rat brain and in radial glial cultures. Glia 15, 157-166.

Sawhney, N., and Hall, P. A. (1992). Ki67--structure, function, and new antibodies. J

Pathol 168, 161-162.

Schafer, M. K., Schutz, B., Weihe, E., and Eiden, L. E. (1997). Target-independent cholinergic differentiation in the rat sympathetic nervous system. Proc Natl Acad Sci

U S A 94, 4149-4154.

Scheer, N., Groth, A., Hans, S., and Campos-Ortega, J. A. (2001). An instructive

function for Notch in promoting gliogenesis in the zebrafish retina. Development 128,

1099-1107.

Schildkraut, J. M., Moorman, P. G., Bland, A. E., Halabi, S., Calingaert, B., Whitaker,

R., Lee, P. S., Elkins-Williams, T., Bentley, R. C., Marks, J. R., and Berchuck, A.

(2008). Cyclin E overexpression in epithelial ovarian cancer characterizes an etiologic

subgroup. Cancer Epidemiol Biomarkers Prev 17, 585-593.

Schmitt, C. A., and Lowe, S. W. (2001). Bcl-2 mediates chemoresistance in matched

pairs of primary E(mu)-myc lymphomas in vivo. Blood Cells Mol Dis 27, 206-216.

182

Schneider, C., Wicht, H., Enderich, J., Wegner, M., and Rohrer, H. (1999). Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons.

Neuron 24, 861-870.

Schnitzer, J., Franke, W. W., and Schachner, M. (1981). Immunocytochemical

demonstration of vimentin in astrocytes and ependymal cells of developing and adult

mouse nervous system. J Cell Biol 90, 435-447.

Schwab, M., Varmus, H. E., and Bishop, J. M. (1985). Human N-myc gene

contributes to neoplastic transformation of mammalian cells in culture. Nature 316,

160-162.

Schwab, M., Westermann, F., Hero, B., and Berthold, F. (2003). Neuroblastoma:

biology and molecular and chromosomal pathology. Lancet Oncol 4, 472-480.

Sheridan, K. M., and Maltese, W. A. (1998). Expression of Rab3A GTPase and other

synaptic proteins is induced in differentiated NT2N neurons. J Mol Neurosci 10, 121-

128.

Shi, H., Cui, H., Alam, G., Gunning, W. T., Nestor, A., Giovannucci, D., Zhang, M.,

and Ding, H. F. (2008). Nestin expression defines both glial and neuronal progenitors in postnatal sympathetic ganglia. J Comp Neurol 508, 867-878.

Shiga, K., Shiga, C., Sasano, H., Miyazaki, S., Yamamoto, T., Yamamoto, M.,

Hayashi, N., Nishihira, T., and Mori, S. (1993). Expression of c-erbB-2 in human

esophageal carcinoma cells: overexpression correlated with gene amplification or

with GATA-3 transcription factor expression. Anticancer Res 13, 1293-1301.

183

Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J., and

Dirks, P. B. (2003). Identification of a cancer stem cell in human brain tumors.

Cancer Res 63, 5821-5828.

Slack, R., Lach, B., Gregor, A., al-Mazidi, H., and Proulx, P. (1992). Retinoic acid-

and staurosporine-induced bidirectional differentiation of human neuroblastoma cell

lines. Exp Cell Res 202, 17-27.

Small, M. B., Hay, N., Schwab, M., and Bishop, J. M. (1987). Neoplastic

transformation by the human gene N-myc. Mol Cell Biol 7, 1638-1645.

Smith, A. (2006). A glossary for stem-cell biology. Nature 441, 1060.

Smith, E., Hargrave, M., Yamada, T., Begley, C. G., and Little, M. H. (2002).

Coexpression of SCL and GATA3 in the V2 interneurons of the developing mouse

spinal cord. Dev Dyn 224, 231-237.

Sommer, L., Shah, N., Rao, M., and Anderson, D. J. (1995). The cellular function of

MASH1 in autonomic neurogenesis. Neuron 15, 1245-1258.

Spengler, B. A., Lazarova, D. L., Ross, R. A., and Biedler, J. L. (1997). Cell lineage

and differentiation state are primary determinants of MYCN gene expression and

malignant potential in human neuroblastoma cells. Oncol Res 9, 467-476.

Spirin, K. S., Simpson, J. F., Takeuchi, S., Kawamata, N., Miller, C. W., and Koeffler,

H. P. (1996). p27/Kip1 mutation found in breast cancer. Cancer Res 56, 2400-2404.

Stanke, M., Junghans, D., Geissen, M., Goridis, C., Ernsberger, U., and Rohrer, H.

(1999). The Phox2 homeodomain proteins are sufficient to promote the development

of sympathetic neurons. Development 126, 4087-4094.

184

Stanke, M., Stubbusch, J., and Rohrer, H. (2004). Interaction of Mash1 and Phox2b in sympathetic neuron development. Mol Cell Neurosci 25, 374-382.

Steenbergen, R. D., OudeEngberink, V. E., Kramer, D., Schrijnemakers, H. F.,

Verheijen, R. H., Meijer, C. J., and Snijders, P. J. (2002). Down-regulation of GATA-

3 expression during human papillomavirus-mediated immortalization and cervical

carcinogenesis. Am J Pathol 160, 1945-1951.

Stemple, D. L., and Anderson, D. J. (1992). Isolation of a stem cell for neurons and

glia from the mammalian neural crest. Cell 71, 973-985.

Stenmark, H., and Olkkonen, V. M. (2001). The Rab GTPase family. Genome Biol 2,

REVIEWS3007.

Stephenson, J. L., and Byers, M. R. (1995). GFAP immunoreactivity in trigeminal

ganglion satellite cells after tooth injury in rats. Exp Neurol 131, 11-22.

Stiles, B., Groszer, M., Wang, S., Jiao, J., and Wu, H. (2004). PTENless means more.

Dev Biol 273, 175-184.

Sudhof, T. C. (1995). The synaptic vesicle cycle: a cascade of protein-protein

interactions. Nature 375, 645-653.

Sugimoto, T., Mine, H., Horii, Y., Takahashi, K., Nagai, R., Morishita, R., Komada,

M., Asada, Y., and Sawada, T. (2000). Neuroblastoma cell lines showing smooth

muscle cell phenotypes. Diagn Mol Pathol 9, 221-228.

Sugimoto, T., Ueyama, H., Hosoi, H., Inazawa, J., Mine, H., Matsumura, T., Fushiki,

S., Kato, T., Miyake, M., and Sawada, T. (1991). [Human neuroblastoma cell lines

having smooth muscle cell markers]. Hum Cell 4, 248-256.

185

Sutherland, H. J., Blair, A., and Zapf, R. W. (1996). Characterization of a hierarchy in

human acute myeloid leukemia progenitor cells. Blood 87, 4754-4761.

Svensson, E. C., Huggins, G. S., Dardik, F. B., Polk, C. E., and Leiden, J. M. (2000a).

A functionally conserved N-terminal domain of the friend of GATA-2 (FOG-2)

protein represses GATA4-dependent transcription. J Biol Chem 275, 20762-20769.

Svensson, E. C., Huggins, G. S., Lin, H., Clendenin, C., Jiang, F., Tufts, R., Dardik, F.

B., and Leiden, J. M. (2000b). A syndrome of tricuspid atresia in mice with a targeted

mutation of the gene encoding Fog-2. Nat Genet 25, 353-356.

Takeuchi, S., Koeffler, H. P., Hinton, D. R., Miyoshi, I., Melmed, S., and Shimon, I.

(1998). Mutation and expression analysis of the cyclin-dependent kinase inhibitor

gene p27/Kip1 in pituitary tumors. J Endocrinol 157, 337-341.

Teitz, T., Wei, T., Valentine, M. B., Vanin, E. F., Grenet, J., Valentine, V. A., Behm,

F. G., Look, A. T., Lahti, J. M., and Kidd, V. J. (2000). Caspase 8 is deleted or

silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nat

Med 6, 529-535.

Tevosian, S. G., Albrecht, K. H., Crispino, J. D., Fujiwara, Y., Eicher, E. M., and

Orkin, S. H. (2002). Gonadal differentiation, sex determination and normal Sry

expression in mice require direct interaction between transcription partners GATA4

and FOG2. Development 129, 4627-4634.

186

Tevosian, S. G., Deconinck, A. E., Tanaka, M., Schinke, M., Litovsky, S. H., Izumo,

S., Fujiwara, Y., and Orkin, S. H. (2000). FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels

from epicardium. Cell 101, 729-739.

Thomson, S. P., and Meyskens, F. L., Jr. (1982). Method for measurement of self-

renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. Cancer Res 42, 4606-4613.

Tong, Q., Tsai, J., Tan, G., Dalgin, G., and Hotamisligil, G. S. (2005). Interaction

between GATA and the C/EBP family of transcription factors is critical in GATA-

mediated suppression of adipocyte differentiation. Mol Cell Biol 25, 706-715.

Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994). mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8,

1224-1234.

Toyoshima, H., and Hunter, T. (1994). p27, a novel inhibitor of G1 cyclin-Cdk

protein kinase activity, is related to p21. Cell 78, 67-74.

Triolo, D., Dina, G., Lorenzetti, I., Malaguti, M., Morana, P., Del Carro, U., Comi, G.,

Messing, A., Quattrini, A., and Previtali, S. C. (2006). Loss of glial fibrillary acidic

protein (GFAP) impairs Schwann cell proliferation and delays nerve regeneration

after damage. J Cell Sci 119, 3981-3993.

Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L., and Jacks, T. (1998).

Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends

survival of Rb-deficient mouse embryos. Mol Cell 2, 293-304.

187

Tsang, A. P., Fujiwara, Y., Hom, D. B., and Orkin, S. H. (1998). Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG. Genes Dev 12, 1176-1188.

Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Yu, C., Weiss, M. J.,

Crossley, M., and Orkin, S. H. (1997). FOG, a multitype zinc finger protein, acts as a

cofactor for transcription factor GATA-1 in erythroid and megakaryocytic

differentiation. Cell 90, 109-119.

Tsarovina, K., Pattyn, A., Stubbusch, J., Muller, F., van der Wees, J., Schneider, C.,

Brunet, J. F., and Rohrer, H. (2004). Essential role of Gata transcription factors in

sympathetic neuron development. Development 131, 4775-4786.

Usary, J., Llaca, V., Karaca, G., Presswala, S., Karaca, M., He, X., Langerod, A.,

Karesen, R., Oh, D. S., Dressler, L. G., et al. (2004). Mutation of GATA3 in human

breast tumors. Oncogene 23, 7669-7678.

Usui, T., Nishikomori, R., Kitani, A., and Strober, W. (2003). GATA-3 suppresses

Th1 development by downregulation of Stat4 and not through effects on IL-12Rbeta2

chain or T-bet. Immunity 18, 415-428.

van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A. P., et al. (2002). The beta- catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111, 241-250.

van der Wees, J., van Looij, M. A., de Ruiter, M. M., Elias, H., van der Burg, H.,

Liem, S. S., Kurek, D., Engel, J. D., Karis, A., van Zanten, B. G., et al. (2004).

188

Hearing loss following Gata3 haploinsufficiency is caused by cochlear disorder.

Neurobiol Dis 16, 169-178.

van Doorninck, J. H., van Der Wees, J., Karis, A., Goedknegt, E., Engel, J. D.,

Coesmans, M., Rutteman, M., Grosveld, F., and De Zeeuw, C. I. (1999). GATA-3 is

involved in the development of serotonergic neurons in the caudal raphe nuclei. J

Neurosci 19, RC12.

Van Esch, H., Groenen, P., Nesbit, M. A., Schuffenhauer, S., Lichtner, P.,

Vanderlinden, G., Harding, B., Beetz, R., Bilous, R. W., Holdaway, I., et al. (2000).

GATA3 haplo-insufficiency causes human HDR syndrome. Nature 406, 419-422.

van Hamburg, J. P., de Bruijn, M. J., Dingjan, G. M., Beverloo, H. B., Diepstraten, H.,

Ling, K. W., and Hendriks, R. W. (2008). Cooperation of Gata3, c-Myc and Notch in

malignant transformation of double positive thymocytes. Mol Immunol 45, 3085-

3095.

van Noesel, M. M., and Versteeg, R. (2004). Pediatric neuroblastomas: genetic and

epigenetic 'danse macabre'. Gene 325, 1-15.

Varley, J. E., McPherson, C. E., Zou, H., Niswander, L., and Maxwell, G. D. (1998).

Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures. Dev Biol 196,

107-118.

Varnum-Finney, B., Xu, L., Brashem-Stein, C., Nourigat, C., Flowers, D., Bakkour,

S., Pear, W. S., and Bernstein, I. D. (2000). Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med

6, 1278-1281. 189

Viger, R. S., Guittot, S. M., Anttonen, M., Wilson, D. B., and Heikinheimo, M.

(2008). Role of the GATA family of transcription factors in endocrine development,

function, and disease. Mol Endocrinol 22, 781-798.

Walton, J. D., Kattan, D. R., Thomas, S. K., Spengler, B. A., Guo, H. F., Biedler, J. L.,

Cheung, N. K., and Ross, R. A. (2004). Characteristics of stem cells from human

neuroblastoma cell lines and in tumors. Neoplasia 6, 838-845.

Wang, A., Yoshimi, N., Ino, N., Tanaka, T., and Mori, H. (1997). Overexpression of

cyclin B1 in human colorectal cancers. J Cancer Res Clin Oncol 123, 124-127.

Wechsler-Reya, R., and Scott, M. P. (2001). The developmental biology of brain

tumors. Annu Rev Neurosci 24, 385-428.

Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81,

323-330.

Weiss, M. J., and Orkin, S. H. (1995). GATA transcription factors: key regulators of

hematopoiesis. Exp Hematol 23, 99-107.

Weiss, W. A., Aldape, K., Mohapatra, G., Feuerstein, B. G., and Bishop, J. M. (1997).

Targeted expression of MYCN causes neuroblastoma in transgenic mice. EMBO J 16,

2985-2995.

Wetmore, C. (2003). Sonic hedgehog in normal and neoplastic proliferation: insight

gained from human tumors and animal models. Curr Opin Genet Dev 13, 34-42.

Wu, X., and Levine, A. J. (1994). p53 and E2F-1 cooperate to mediate apoptosis. Proc

Natl Acad Sci U S A 91, 3602-3606.

190

Xu, H., Firulli, A. B., Zhang, X., and Howard, M. J. (2003). HAND2 synergistically

enhances transcription of dopamine-beta-hydroxylase in the presence of Phox2a. Dev

Biol 262, 183-193.

Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996).

Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85, 537-548.

Yamashita, M., Shinnakasu, R., Asou, H., Kimura, M., Hasegawa, A., Hashimoto, K.,

Hatano, N., Ogata, M., and Nakayama, T. (2005). Ras-ERK MAPK cascade regulates

GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. J

Biol Chem 280, 29409-29419.

Yang, H. Y., and Evans, T. (1992). Distinct roles for the two cGATA-1 finger

domains. Mol Cell Biol 12, 4562-4570.

Yao, D., Zhang, X., Wei, H., and Tian, Z. (2005). Antisense-induced blockade of

GATA-3 expression could inhibit Th2 excursion of tumor cells in vitro and in vivo.

Cell Mol Immunol 2, 189-196.

Yilmaz, O. H., Valdez, R., Theisen, B. K., Guo, W., Ferguson, D. O., Wu, H., and

Morrison, S. J. (2006). Pten dependence distinguishes haematopoietic stem cells from

leukaemia-initiating cells. Nature 441, 475-482.

Ying, Q. L., Nichols, J., Chambers, I., and Smith, A. (2003). BMP induction of Id

proteins suppresses differentiation and sustains embryonic stem cell self-renewal in

collaboration with STAT3. Cell 115, 281-292.

191

Yuan, Y., Shen, H., Franklin, D. S., Scadden, D. T., and Cheng, T. (2004). In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nat Cell Biol 6, 436-442.

Zhang, S., Balch, C., Chan, M. W., Lai, H. C., Matei, D., Schilder, J. M., Yan, P. S.,

Huang, T. H., and Nephew, K. P. (2008). Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 68, 4311-

4320.

Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. M., Sampath, J., Morris, J. J.,

Lagutina, I., Grosveld, G. C., Osawa, M., Nakauchi, H., and Sorrentino, B. P. (2001).

The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7, 1028-1034.

Zurawel, R. H., Chiappa, S. A., Allen, C., and Raffel, C. (1998). Sporadic medulloblastomas contain oncogenic beta-catenin mutations. Cancer Res 58, 896-899.

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ABSTRACT

Sympathetic ganglia are primarily composed of noradrenergic neurons and

satellite glial cells. Although both cell types originate from neural crest cells, the

identities of the progenitor populations at intermediate stages of the differentiation

process remain to be established. Here we report the identification in vivo of glial and

neuronal progenitor cells in postnatal sympathetic ganglia, using mouse superior

cervical ganglia as a model system. There are significant levels of cellular

proliferation in mouse superior cervical ganglia during the first 18 days after birth. A

majority of the proliferating cells express both nestin and brain lipid-binding protein

(BLBP). BrdU fate-tracing experiments demonstrate that these nestin and BLBP

double positive cells represent a population of glial progenitors for sympathetic

satellite cells. The glial differentiation process is characterized by a marked

downregulation of nestin and upregulation of S100, with no significant changes in the

levels of BLBP expression. We also identify a small number of proliferating cells that

express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis

that defines sympathetic noradrenergic neurons. Together, these results establish

nestin as a common marker for sympathetic neuronal and glial progenitor cells and

delineate the cellular basis for the generation and maturation of sympathetic satellite

cells.

This research in the normal postnatal sympathetic development provides the

basis for the neuroblastoma study. Neuroblastoma is the most common childhood malignant tumor which originates from sympathetic nervous system. 90% of children

193

with this disease are diagnosed before 6 years old. Therefore, the tumorigenesis of

neuroblastoma may represent an abnormal embryonic or postnatal sympathetic

development. Transformed neural crest stem cells or malignant sympathetic precursor

cells that obtain stem cell abilities are recognized as neuroblastoma stem cells that

might be the origin of neuroblastoma. BE(2)-C cells, a human neuroblastoma cell line

enriched with tumorigenic stem cells, can be induced to undergo either neuronal or

glial differentiation. We use BE(2)-C cells as a system to identify the genes that

regulate neuroblastoma stem cell activities, such as self-renewal and differentiation.

One of these genes is GATA3, a zinc-finger transcription factor with an essential role

in sympathetic development. Downregulation of GATA3 by siRNA promotes BE(2)-

C cell proliferation and overexpression of GATA3 decreases the proliferation of

BE(2)-C cells. GATA3 regulates cell proliferation through distinct pathways

involving Cyclin D1 and E2F1, as evidenced by an increased expression of Cyclin D1

and a decreased expression of E2F1 in GATA3 knockdown cells. In addition, GATA3 knockdown induces BE(2)-C cells to undergo glial differentiation, as indicated by an increase in the expression of GFAP, a glial cell marker, and a decrease in the expression of neuronal marker SNAP25. GATA3 overexpression promotes neuronal differentiation of BE(2)-C cells, as indicated by a reduction of GFAP expression and an upregulation of SNAP25 expression. GATA3 knockdown also downregulates

Phox2b and GATA3 overexpression upregulates Mash1, two transcription factors that

are expressed in neuronal progenitor cells and are essential for sympathetic

development. Together, these findings suggest that GATA3, Phox2b and Mash1 may function in a regulatory network in the control of neuroblastoma cells in a stem/progenitor cell state.

194