Dissertation Approved

By

Major Advisor

Philip R. Brauer, Ph.D., Associate Professor of Biomedical

Sciences

Barbara Braden, Ph.D. MATRIX METALLOPROTEINASE ACTIVITY IS

AN IMPORTANT MEDIATOR OF CARDIAC

NEURAL CREST CELL MIGRATION

By

Dong Hong Cai

A DISSERTATION

Submitted to the Faculty of the Graduate School of Creighton

University in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in the Department of Biomedical Sciences

Omaha, Nebraska, December, 2001

II This Dissertation Is Dedicated to:

My Dear Wife Hong Tao and My Coming Son

III Acknowledgments

I would like to express my sincere acknowledgment to my major advisor, Dr.

Philip R. Brauer, for his guidance, encouragement, and patience throughout all these years. His compassion for science and thirst for knowledge has deeply impressed me.

I would like to thank my committee members Dr. John A. Yee, Dr. Robert

Mackin, and Dr. Margaret scofield, for their guidance via numerous discussions and for their generosity to let me use their facilities.

I would like to thank my former committee member Dr. Thomas M. Vollberg who trained me in molecular biology and generously provided me his lab facilities during my first two years in this department.

I would like to thank Dr. James P. Quigley for providing MMP-2 and TIMP-2 cDNA used in this program.

1 would like to thank my parents and my wife for their support and love over the years.

Finally, I would like to thank all my friends and colleagues in this department who provided numerous helps whenever help was needed.

IV ABSTRACT

Matrix metalloproteinases (MMPs) are a family of zinc proteolytic enzymes important in embryonic development and pathological processes. Cardiac

(CNC) cells are derived from the dorsal portion of through an epithehal-to - mesenchymal cell transition and then migrate along the basement membrane of , enter the pharyngeal arches, and subsequently participate in the septation of the heart during embryonic development. My studies showed that one of the MMPs, MMP-2

(gelatinase A, or 72 kDa type IV collagenase), was deposited in CNC migration pathway and was expressed by CNC cells after they entered the pharyngeal arches. Furthermore, the distribution pattern of MMP-2 was disrupted by migrating CNC cells. This suggested that MMP enzymatic activity might play an important role in CNC migration. To test this hypothesis, a synthetic MMP inhibitor KB8301 was injected into the cell-free space adjacent to the premigratory CNC cells. The distance that CNC migrated in the injected side was significantly decreased compared with that in the noninjected side and in the vehicle treated embryos. Enzyme activity assays showed that the activity of MMPs decreased about 30% after KB8301 injection. This demonstrated that enzymatic activity of MMPs is an important mediator for CNC migration. The natural inhibitors of MMPs,

TIMPs, were also studied. TIMP-2, which not only has the inhibitory activity against

MMPs, but also participates in the activation of proMMP-2, was expressed exclusively in a subpopulation of CNC cells during their early migration. Local delivery of exogenous

TIMP-2 significantly decreased CNC migratory distance as well as MMP activity in vivo.

The pattern of TIMP-3 expression suggests that this inhibitor is not likely to have a role in early NC migration but could be involved in pharyngeal arch and cardiac development.

V Overall, my studies support the hypothesis that MMP activity is an important mediator of

CNC cell migration.

VI TABLE OF CONTENTS

Dedication III

Acknowledgments IV

Abstract V

Table of Contents VII

List of Tables and Figures IX

Chapter One: General Introduction and Background 1

Chapter Two: MMP-2 expression During Early Avian

Morphogenesis 17

Introduction 18

Materials and Methods 21

Results 24

Discussion 28

Chapter Three: A synthetic MMP Inhibitor Decreases Early Cardiac Neural

Crest Migration in Chicken Embryos 40

Introduction 41

Materials and Methods 44

Results 47

Discussion 51

Chapter Four: Three Role of TIMP-2 and TIMP-3 During Early Cardiac

Neural Crest Formation and Migration 71

Introduction 72

Materials and Methods 76

VII Results 79

Discussion 82

General Discussion 100

Summary 104

References 105

Appendix 128

VIII LIST OF TABLES AND FIGURES

Chapter One

Table 1-1. Timetable for CNC Migration 14

Table 1-2. Nomenclature, Natural Substrates and Inhibitors of the 15

MMPs

Figure l-l. Model of cell surface activation of proMMP-2 16

Chapter Two

Figure 2-1. MMP-2 and MT3-MMP mRNA expression during early

neural crest moiphogenesis 33

Figure 2-2. Image of a gelatinase zymogram prepared using equal

amounts of protein from tissue lysates of whole mount embryos of

stage 9/10 (A) and stage 13/14 (B) 36

Figure 2-3. MMP-2 immunostaining 38

Chapter Three

Table 3-1. Effect of KB8301 Injection on MMP Activity 58

Table 3-2. Effect of KB8301 Injection on Early Embryonic 59

Development

Table 3-3. Effect of KB8301 on CNC cell Migration When

IX Normalized for Embryo Size 60

Table 3-4. Relationship Between CNC Migratory Distance and

Developmental Stage at the Time of Collection 61

Figure 3-1. Dorsal view of whole mount FINK-1 immunostained

embryos sham operated, treated with DMSO, or treated with 62

KB 8301

Figure 3-2. DAPI nuclear staining pattern at the 2nd somite level in

DMSO-treated and KB 8301-treated embryos 62

Figure 3-3. Measuring CNC migratory distance in DMSO-treated

and KB8301-treated (92 pmoles) embryos 62

Figure 3-4. Effect of 46 pmoles of KB8301 on CNC cell migratory

distance when injected unilaterally into embryos at stages 10‘, 10, or 65

10+

Figure 3-5. Effect of 92 pmoles of KB8301 on CNC cell migratory

distance when injected unilaterally into embryos at stages 10', 10, or 67

10+

Figure 3-6. Cross-sectional area of the neural tube in embryos

treated unilaterally with DMSO or 92 pmoles of KB8301 measured

at the level of the 2nd somite 69

Chapter Four

Table 4-1. Effect of TIMP-2 Injection on MMP Enzymatic Activity 90

Table 4-2. Effect of TIMP-2 Injection on Embryonic Development 91

X Table 4-3. Effect of TIMP-2 Injection on CNC Migration After

Normalizing for Potential Differences in Embryo Size 92

Figure 4-1. TIMP-2 and TIMP-3 mRNA expression during early

CNC morphogenesis 93

Figure 4-2. DAPI nuclear staining of sections from Ringer’s-treated and TIMP-2-treated embryos 96

Figure 4-3. Measurement of CNC migratory distance on cross sections from Ringer’s-treated or TIMP-2-treated embryos 96

Figure 4-4. Effect of TIMP-2 injection on CNC cell migration when injected unilaterally into embryos at stage 10 98

XI CHAPTER ONE

GENERAL INTRODUCTION AND BACKGROUND

l A. Chicken Cardiogenesis

During gastrulation, mesodermal cells in the early chicken embryo specific to heart lineage emerge from the epithelial epiblast of the rostral half of the primitive streak [1],

These mesodermal cells migrate anteriorly and ventrally along the endoderrnal basement membrane and form the paired primordia of the embryonic heart [2J. By Hamburger &

Hamilton (HH) stage 6 [3], these primitive cells segregate and form two lineages, the endocardial and myocardial lineage [4].

The precardiac mesoderm of the paired primordia fuse to form a single straight tubular heart at the ventral midline by late HH stage 8 to early stage 9. This tubular heart consists of an outer myocardial layer separated from the endocardium by an extracellular matrix (ECM) called cardiac jelly. Thereafter, structural changes occur within the developing heart essential to its normal development. First, the heart undergoes looping, transforming it into a S-shaped tube. The convexity of the loop demarcates the functional inflow from an outflow portion of heart. Convergence of the inflow and outflow tracts follows while the primitive heart chambers expand. As the chambers expand, extensive remodeling aligns the right and left atrioventricular (AV) canals with future left and right ventricles, reduces the size of the bulboventricular flange, and widens of the conus arteriosus. Later, several septa form simultaneously which separate the ventricle and atrium, and divide the primitive ventricle and atrium into left and right sides. In the meantime, a process called wedging adjusts the position of the outflow tract so the future aortic side of the outflow tract is nestled between the mitral and tricuspid valves. Outflow tract septation occurs concurrently with the wedging process so that the forming

2 aorticopulmonary septum is in the correct position and that this septum converges with the interventricular septum and the forming AV septum.

In addition to sculpting events, successful septation of the heart also requires the formation and migration of two distinct mesenchymal populations into the cardiac jelly.

One population forms through an epithelial-mesenchymal cell transition of the atrioventricular and outflow tract endocardium and the other is derived from the invading neural crest (NC) cells.

B. Cardiac Neural Crest Cells

The neural crest cells arise from the junction between ectoderm and . As the neural plate closes to form the neural tube, NC cells are released from the neural folds

[9]. Cardiac NC (CNC) cells are derived from the dorsal part of the neural tube between the mid-otic level and caudal end of the somite 3. These cells begin to segregate from neural epithelial cells at stage 9 and start to migrate out of the epithelium around stage

10. A timetable for CNC cell migration is shown in Table 1-1. CNC cells migrate dorsalaterally under the to reach the circumpharyngeal region, an area adjacent to the pharyngeal pouches. CNC cells derived from the neural tube between the midotic and rostral end of somite 1, somite 1 and somite 2, and caudal end of somite 2 and caudal end of somite 3, migrate into pharyngeal arches 3, 4, and 6 respectively. Here one subpopulation of CNC cells contribute to the formation of the tunica media of the aortic arch arteries [11], Another subpopulation of CNC cells migrate into the outflow tract and participate in forming the aorticopulmonary septum [12]. Although all of the

CNC cells in the pharyngeal arches 3, 4, and 6 contribute to outflow tract septation, those

3 from pharyngeal arch 4 provide four times as many cells as those from arch 3 and 6 [13].

Most of the CNC cells emigrating from the neural folds come to populate the dorsal cushion in the truncus arteriosus and come to lie adjacent to its endocardium. Some CNC

cells in this group continue to migrate further into the conus arteriosus and contribute to

the septation of this segment of the outflow tract as well. In addition, CNC cells

contribute to the formation of the two semilunar valves, the membranous portion of the

interventricular septum, the wall of the pulmonary infundibulum, and transiently within

the wall of the aortic vestibule [12]. Therefore, CNC cells play an integral role in the

normal development of the heart.

Several human congenita! abnormalities and mouse mutations have been linked to

defects in NC differentiation or migration. In human, patients with DiGeorge Syndrome

or velocardiofacial syndrome exhibit broad and variable phenotypes that include

conotruncal cardiac defects, hypocalcemia, and palatal and facial anomalies, all of which

appear to be linked by NC dysgenesis. Although these defects have been ascribed to the

deletions of chromosomal region 22ql 1.2, the molecular mechanisms are poorly

understood. In mouse, Pax3 mutants, which over-express the chondroitin sulphate

proteoglycan, versican, and Patch mutants that are missing the normal alpha subunit of

the platelet-derived growth factor, result in defective NC migration and similar

developmental defects as seen in patients with DiGeorge Syndrome [1,2].

C. MMPs

Matrix metalloproteinases (MMPs) are an important family of zinc enzymes

responsible for degradation of the extracellular matrix components during normal

4 embryogenesis and tissue remodeling as well as in many disease processes [3-5], This superfamily has more than 20 members and is divided into four groups: collagenases, strornelysins, gelatinases, and membrane-type MMPs (MT-MMP). All the MMPs are secreted in a latent zymogen form and share a common domain structure composed of propeptide, catalytic, hinge, and hemopexin-like domains, except MMP-7 (matrilysin) which lacks the hemopexin-like domain [6] and MT-MMPs which have an extra transmembrane domain at the C terminus and are intercalated into the plasma membrane

[7].

The catalytic mechanism of MMPs depends on the presence of zinc at their active centers (the conserved HEXGHX[L/M]G[L/M]XH motif is responsible for ligating zinc in the catalytic domain). In proMMPs, the 80-amino-acid-long propeptide contains a conserved sequence PRCG[V/N]PDV. The cysteinyl residue in this sequence interacts with the catalytic Zn2+ of the active site preventing the Zn2+ from associating with water thereby keeping the zymogen inactive. Activation of MMP zymogens requires proteolytic removal of this amino-terminal propeptide by active MMPs, serine-proteinases, or MT-

MMPs [8], Once removed, Zn"+ binds and activates a water molecule allowing it to attack

the peptide bond of the MMP substrate. In most situations, the propeptide-free enzyme needs further processing to be fully active and become more stable [9]. For example, the

72 kDa form of proMMP-2 is converted to a 66 kDa intermediate form after the removal

of propeptide. This intermediate continues to be autolytically processed into a 62 kDa

mature active MMP-2. In contrast, MT-MMPs are activated intraceliularly by Golgi-

associated enzymes of furin family [8]. Every MMP has more than one substrate, which

is summarized in the Table 1-2 [8],

5 The enzyme activity of MMPs is inhibited by tissue inhibitors of MMPs (TIMPs), which have four members named TIMP-1, TIMP-2, TIMP-3, and TIMP--4.

Intermolecular binding between MMP and TIMP is mediated via the carboxyl-terminal, hemopexin-like domain of the MMPs and the non-inhibitory carboxyl-terminal domain of the TIMP molecule. This binding aligns these two molecules and facilitates the inhibitory activity of the amino-terminal domain of the TIMP when the MMP becomes active [10].

The degree of MMP activity depends on the ratio between MMPs and TIMPs. If the amount of active MMP molecules is more than that of TIMP, MMPs will degrade their substrates, otherwise the activities of MMPs are quenched by TIMPs. TIMPs not only control the proteolytic activity of MMPs but also may regulate their processing from zymogen to active forms. For example, TIMP-2 is essential for the activation of proMMP-2 (discussed below).

Among all the MMPs, MMP-2 has been widely studied because it can degrade basement membrane components and is directly involved in malignant cell metastasis

[11,12], endothelial cell migration [13], and epithelial cell migration [14]. Data show that these cells express MMP-2 or recruit MMP-2 to the cell surface through MT-MMPs and then remodel the surrounding ECM [15,16].

The activation process of proMMP-2 is distinct from those of other MMP zymogens.

Many proteinases capable of activating other proMMPs are relative poor activators of proMMP-2 [17]. The primary activators of proMMP-2, the MT-MMPs, including MT1-

MMP, MT2-MMP, and MT3-MMP [9], Several studies suggest that cell surface MT-

MMP binds to TIMP-2 and the MT-MMP/TIMP-2 complex then recruits extracellular proMMP-2 to cell surface through the binding between TIMP-2 and proMMP-2. Once

6 bound, the proMMP-2 is activated by neighboring unoccupied MT-MMP (Fig. 1-1).

Further evidence that the principle pathway for proMMP-2 activation is mediated by

TIMP-2 is shown by the observation that no detectable active MMP-2 is found in TIMP-2

null mice [18]. Other TIMPs, like TIMP-1 and TIMP-3, also inhibit the activity of MMP-

2. Like TIMP-2, TIMP-1 and TIMP-3 are widely co-expressed with MMP-2 during embryogenesis, tissue remodeling, and tumor metastasis and are sometimes as important

as TIMP-2 in inhibiting MMP-2 activities [19] [20],

D. MMPs Are Required In Cell Migration and Tissue Remodeling

It is well established in the literature that MMPs can regulate malignant cell migration. For example, Ray et. al. (1995) demonstrated that inhibition of endogenous

MMP-2 activity in melanoma cells, which are derivatives of NC cells, resulted in not only decreased proteolysis of the ECM, but also enhanced the adhesion and spreading properties of these cells, properties of the non-metastasizing cells [12]. Furthermore,

Nakahara et. al. (1997) detected specific localization of MT1-MMP within the invadopodia of melanoma cells where proMMP-2 was recruited and activated to facilitate

ECM degradation and cell invasiveness [21], In histological specimens from human melanocytic tumors, Vaisanen et. al. (1996) immunolocalized MMP-2 in melanocytic lesions but not in surrounding normal skin. The number of MMP-2-positive cells increased with decreasing architectural organization and increasing atypia in the melanocytic lesions. In addition, the MMP-2 positivity in the primary and subcutaneous melanoma lesions correlated with later hematogenous metastasis [22]. In an animal model, Schultz et. al. (1998) showed that an intraperitoneal injection of recombinant

7 TTMP-l reduced lung colonization of intravenously injected melanoma cells [23]. Similar observations were made by using synthetic inhibitors against MMPs in experimental metastasis assays of melanoma, mammary carcinoma, and colorectal tumor cells [24-26],

These results provide strong support for a role for MMPs in establishing metastatic lesions. This is an active area of study because of therapeutic application to treatment of cancer.

In normal cell lines, MMP-2 and MMP-9 have been found to be localized into the basolateral plasma membrane or focal contacts in bovine pulmonary microvascular endothelial cells [27]. Focal contacts contain integrins that are associated with the actin cytoskeleton and bind extracellular components like collagens and fibronectins. Forming and disrupting focal contact is a key step in endothelial cell migration. In the initial stages of capillary formation (angiogenesis), microvascular endothelial cells of preexisting blood vessels locally degrade the underlying basement membrane and invade the stroma of the tissue to be vascularized. Microvascular endothelial cells express MMP-2 and

MT1-MMP and form endothelial cell networks in three-dimensional collagen lattices

[13]. Treatment of the cultures with MMP inhibitors block the activities of MMPs and inhibit formation of the endothelial cell networks. Transfection of MT1-MMP into invasion-incompetent endothelial cells also induces their penetration through fibrin barriers [28]. Furthermore, application of the hemopexin-like domain of MMP-2 involved in MMP-2 binding via a v(33 inlegrin blocks MMP-2 activity and disrupts angiogenesis

[29], These studies show that MMPs may initiate and promote cell invasion and migration in normal cells.

8 MMPs are also involved in tissue remodeling. During angiogenesis, the basement membrane is degraded and stroma of the underlying tissue to be vascularized is remodeled by MMPs. During munne embryogenesis, Chin et. al. (1997) detected the expression of MMP-2, MMP-9, and other MMPs, as well as their inhibitors TIMP-1,

TIMP-2, TIMP--3 in the mandibular arch. By applying a synthetic inhibitor to mandibular explants, they found that formation of the oral sulcus and fusion of the two epithelia of the medial sulcus were inhibited. In addition, the development of the anterior segment of

Meekers cartilage was disrupted and number and migration of myoblasts decreased [3].

These data suggest that MMPs regulate cell migration and morphogenesis of structures derived from ectoderm (oral sulcus), somitic mesoderm (tongue), and cranial NC

(Meckel's cartilage).

E. Points Leading to My Hypothesis

CNC cells emigrate from the dorsal portion of neural tube, migrate into pharyngeal

arches, and a subset finally enters the outflow tract of heart. At the beginning of this long journey, CNC cells lose N-cadherin, E-cadherin and N-CAM, the cell adhesion molecules

that secure CNC cells to the neural tube [30,31]. After emigrating from the neural tube,

CNC cells interact with, and migrate within ECM. With respect to NC migration, ECM

can be divided into two groups, the permissive and the non-permissive ECM. The

permissive ECM includes fibronectin, laminin and collagen types I, II and IV, while the

non-permissive molecules include aggrecan (which carries different keratan-sulphate side

chains and carbohydrate moieties) and versican (a member of the chondroitin sulphate

proteoglycan family) [1]. NC cells express cell surface molecules capable of interacting

9 with ECM. Most of these molecules are variant types of integrins (oq family, (3, family, a vPj, a vP3, a vP5 etc.) that mediate NC attachment to the substratum [32-34], However, adhesion of NC cells to a permissive substratum like laminin and fibronectin is not sufficient for their migration. These molecules must be remodeled to release cell adhesion so that NC cells can move [35].

The factors contributing to NC motility are not very clear, but one possibility is that proteases produced by cells may permit them to degrade and therefore break adhesions with substratum proteins [36] or to breach a ECM barriers [37], just as malignant cells and endothelial cells do during their invasion. One of the proteases, the urokinase-type plasminogen activator (uPA), regulates cranial NC cell migration through activation of plasminogen in vitro [38], uPA is also expressed in trunk NC cells in vivo, and may be necessary for trunk NC migration in vitro [39], Interestingly, when the synthetic MMP inhibitor 1,10-phenanthroline is applied to cultured avian trunk NC cells, NC migration decreases [39], In addition, zymograms show avian CNC cells express MMP-2 in vitro

(Brauer, unpublished data). These data suggest the possibility that MMPs are involved in avian CNC migration in vivo.

In avian model, MMP-2 has been detected in developing hearts using zymography,

RT-PCR, and in situ hybridization [40]. MMP-2 mRNA expression in heart is found to remain fairly constant or increases only slightly between stages 12 and 24 of development.

Based on zymography, the 72 kDa proMMP-2 protein level remained constant from stage

12 to 24, while levels of the 66 kDa active MMP-2 form significantly increased from stage

12 to 24. In addition, mRNA transcripts of one of proMMP-2 activators, MT3-MMP, are also increased in parallel suggesting that proMMP-2 activation has a role in heart

10 development. Furthermore, studies show that a synthetic MMP inhibitor blocks endocardial-derived cushion cell development and migration [41]. These data demonstrate

MMPs are expressed in early developing avian embryos and suggest they may be involved in epithelial-mesenchymal transition, cell migration and invasion.

Again in the avian model, ablation of premigratory CNC cells results in so-called

CNC-related abnormalies, including persistent truncus arteriosus, double-outlet right ventricle, tetralogy of Fallot, double-inlet left ventricle, tricuspid atresia, straddling tricuspid valve, and the absence of a varying combination of aortic arch arteries derived from pharyngeal arches 3, 4 (interrupted aortic arch), and 6 (absent ductus arteriosus) [42],

Several types of MMP have been knocked out in the mouse model and none of these abnormalies has been reported. Most of the mutants (MMP-3, MMP-7, MMP-9, MMP-11,

MMP-12 null mice) have normal phenotypes [43-49], MMP-2 knockout mice develop normally and are fertile. According to the authors, these mutant mice do not show any gross anatomical abnormalities but seem to be smaller at birth and show significantly slower growth rates from postnatal day 3 to adulthood. The reason for this is unknown.

While cultured fibroblasts from mutant embryos grow and migrate normally on collagen- coated or gelatin-coated and uncoated dishes, malignant cell invasion, metastasis, and tumor-induced angiogenesis decrease significantly in these animals compared with their normal littermates [50]. MMP-9 knockout mice are also viable, fertile, and show no CNC- related abnormalities. Flowever, MMP-9 mutant mice exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. But 3 weeks after birth, aberrant apoptosis, vascularization, and ossification compensate for this so that the mice acquired a normal appearance [44]. MT1-MMP null mice show craniofacial dysmorphism, arthritis,

11 osteopenia, dwarfism, fibrosis of soft tissues, and early death due to ablation of an essential collagenolytic activity although no CNC-specific defects are reported [48],

MMPs, at least MMP-2 or MMP-9, seem not to be required for CNC cell migration.

However, in MMP-2 knockout mice, the loss of MMP-2 activity in CNC cells could be compensated by other MMPs. As mentioned above, the developmental sequence of the long bone in MMP-9 null animals is abnormal but is not apparent in older animals. It is unknown whether the developmental sequence of CNC cell migration and heart formation in MMP-2 and MMP-9 null mice is different. Therefore, an integral role of

CNC morphogenesis is difficult to exclude using MMP knockout models.

F. Hypothesis and specific aims.

The major aim of my experiments in this thesis was to examine the functional role of

MMPs in CNC cell migration. The hypothesis tested was:

MMP activity is an important mediator of CNC cell migration and inhibiting

MMP activity will decrease CNC cell migration.

The specific aims were:

1. To determine the spatial and temporal distribution of two of the MMPs,

MMP-2 and MT3-MMP, in chick embryos and to determine if their

expression correlated with early CNC cell migration.

2. To determine if a synthetic MMP inhibitor, KB8301, inhibited CNC cell

migration in vivo.

12 3. To determine the spatial and temporal expression of two TIMPs, TIMP-2 and

TIMP-3, in chicken embryos and to determine if their expression correlated

with early CNC cell migration.

4. To determine if injection of exogenous TIMP-2 inhibited CNC cell migration

in vivo.

In this thesis, Chapter 2 describes the spatial and temporal distribution of MMP-2 mRNA and protein, and MT3-MMP mRNA, during early chick CNC morphogenesis and migration. The results showed that MMP-2 protein was deposited in the CNC migration pathway and that its distribution pattern was altered in areas occupied by CNC cells suggesting enzymatic activity of MMP-2 played a role in CNC cell migration. In Chapter

3, a synthetic MMP inhibitor, KB8301, was used to examine if inhibiting the enzymatic activity of MMPs could decrease CNC cell migration in vivo. Results showed that

KB8301 significantly decreased the distance CNC migrated from the neural tube, showing MMP activity is an important mediator of CNC migration in vivo. Finally,

Chapter 4 explored the spatial and temporal expression of two natural MMP inhibitors,

TIMP-2 and TIMP-3, in chicken embryos. Results showed that TIMP-2 was specifically expressed within CNC cells. Hence, recombinant TIMP-2 was also injected into CNC migration pathways to determine if CNC cell migration was reduced.

Collectively, the results of my experiments support the hypothesis that MMP activity is an important mediator of CNC cell migration.

13 Table 1-1

Time Table for CNC Migration

Stage Location 8-9 Segregation of presumptive CNC cells within the dorsal part of neural tube between mid-otic level and caudal end of somite 3 10-11 Emigration of CNC cells from the neural tube and migration under the ectoderm toward the circumpharyngeal area 12-13 CNC cells reach the circumpharyngeal area 14-18 CNC migration temporally ceases within the lateral body wall and then continues into pharyngeal arch 3, 4 and 6 as these pharyngeal arches form 19-20 CNC cells reach the aortic sac at the distal end of the outflow' tract 21-26 CNC cells migrate into the truncus arteriosus w'hile proliferating and forming a condensed aorticopulmonary septum 27-29 CNC cells reach the superior atrioventricular cushion and participate in conal and ventricular septation

34 Table 1-2

Nomenclature, Natural Substrates and Inhibitors of the MMPs

Group MMP/TIMP Enzyme Molecular Molecular weight Known substrates related with this study weight (pro­ (active form) form) Collagenases MMP-1 Collagenase-1 55,000 45,000 Collagens I, II, III, VII, VIII. X; gelatin, aggrecan, versican, proteoglycanlink protein; casein; nidogen; proTNF; L- selectin; MMP-2; MMP-9

MMP-8 Neutrophil 75,000 58,000 Collagens I, II, ill, V, VII, VIII, X; collagenase gelatin; aggrecan; fibronectin MMP-13 Collagenase-3 60,000 48,000 Collagens I, II, III, IV; gelatin; aggrecan; perlecan; tenascir, MP-18 Xenopus 55,000 ? collagenase Stromelysins MMP-3 Stromelysin-1 57,000 45,000 Collagens III, IV, IX, X; gelatin; aggrecan; versican; perlecan; fibronectin; laminin; elastin; casein; MMP-1, -7; -8; -9; -13

MMP-10 Stromelysin-2 57,000 44,000 Collagens HI, IV, V; gelatin; casein; aggrecan; elastin; fibronectin; MMP-1, -8 MMP-11 Stromelysin-3 51,000 44,000 MMP-19 ? 7 MMP-20 enamelysin ? ? amelogenin Gelatinases *M M P-2 72 kDa 72,000 62,000 MMP-9, MMP-13, collagens I, IV, V, gelatinase VII, X, XI, XIV; gelatin, elastin, vitronectin, tenascin, fibronectin, laminin-5, aggrecan, versican *M M P-9 92 kDa 92,000 86,000 collagens IV, V, VII, X, XIV; gelatin; gelatinase elastin; aggrecan; versican; fibronectin; nidogen Membrane type MMP-14 MT1-MMP 66,000 56,000 MMP-2, MMP-13, collagens 1,11,111, M M Ps gelatin, elastin, fibronectin, laminin MMP-15 MT2-MMP 72,000 7 MMP-2, gelatin, fibronectin, laminin *M M P-16 M T 3-M M P 64,000 52,000 M M P-2 MMP-17 MT4-MMP ? ? MT5-MMP ? 7 MMP-25 MT6-MMP ? 7 Tissue inhibitors of TIMP-1 28,500 All MMPs except MMP-14, MMP-19. M M Ps (glycosylated) Binds to proMMP-9 *T IM P-2 21,000 All MMPs. Binds to proMMP-2 (unglycosylated) *T IM P-3 27.000 All MMPs. Binds to proMMP-2 and (glycosylated) proM M P-9 24.000 (unglycosylated) TIMP-4 23,000 MMP-1, 2, 3, 7, 9. Binds to proMMP-2 (unglycosylated) * Chicken DNA sequence known.

15 MT-MMP TIMP-2

MT-MMP proMMP-2

Figure 1-1. Model of cell surface activation of proMMP-2. Step 1: the catalytic domain of membrane-bound MT-MMP binds with the N-terminal domain of TIMP-2 forming MT-MMP/TIMP-2 complex. Step 2: the complex recruits proMMP-2 to cell surface from ECM forming MT-MMP/TIMP- 2/proMMP-2 ternary complex through the interaction between the C-terminal domain of TIMP-2 and the C-terminal domain of proMMP-2. Step 3: neighboring unoccupied MT- MMP activates proMMP-2 by proteolytically removing the propeptide domain from proMMP-2.

16 CHAPTER TWOf

MMP-2 EXPRESSION DURING EARLY AVIAN CARDIAC

NEURAL CREST MORPHOGENESIS

fPortion of this work has been previously published (Cai et. al., Anatomical Record

259:168-179,2000)

17 INTRODUCTION

During embryonic development, septation of the cardiac outflow tract and subsequent formation of the great vessels involve two distinct mesenchymal populations.

One population is derived from the endocardium through an epithelial-to-mesenchymal cell transition [51-53]; the other population arises from the migrating neural crest (NC) cells [54,55], NC cells are also derived from an epithelial-to-mesenchymal transition at the junction between the ectodermal and neural epithelium during [56]. As the neural folds close to form the neural tube, NC cells emigrate from the epithelium and migrate toward a variety of destinations where they participate in the embryonic organogenesis [57], NC cells originating along the dorsal neural tube between mid-otic and third somite level begin emigrating at Hambuger-Hamilton stage 10 in the chick, migrate along the basement membrane of ectoderm, and enter the pharyngeal arches where they are instrumental in the remodeling and development of the great arteries [58-

60]. A subpopulation of these NC cells then enter the outflow tract where they form the core of two spiraling ridges extending toward the ventricle. These ridges then fuse, separating the distal outflow tract into the aorta and pulmonary artery [54,55,61,62],

Hence, NC cells derived from the axial level between mid-otic and 3rd somite are collectively dubbed cardiac NC (CNC) cells although only a subset of these cells will actually enter the heart.

Cell migration and tissue remodeling are two fundamental processes of embryogenesis. Matrix metalloproteinases (MMPs) have been shown to be involved in cell migration and ECM degradation during development. MMPs, particularly the

18 gelatinases/collagenases, are implicated in mediating tissue remodeling and enabling cell

migration and invasiveness [3-5,63], Two forms of gelatinases/collagenases have been extensively studied in cells. Matrix metalloproteinase-type 2 (MMP-2), or gelatinase A, is

secreted as a 72 kDa molecule and is reduced to a 62-64 kDa protein when processed into its active form; matrix metalloproteinase-type 9 (MMP-9), or gelatinase B, is secreted as a 92 kDa molecule and is reduced to a 82-84 kDa protein when processed to its active form. Both can degrade type-IV collagen and interstitial collagens as well as many other

ECM molecules [63-66]. ProMMPs are secreted by cells and can be activated either autolytically, by plasmin, or by membrane-type MMPs (MT-MMPs) [8,65], MT-MMPs are transmembrane MMPs that govern many of the functional activities attributed to extracellular MMPs in addition to their direct proteolysis activity toward ECM components [9,67], In vitro, migration of trunk NC cells and NC-derived mesenchyme is decreased by MMP inhibitors [39,68], Patch mutant mice, which have a chromosomal deletion resulting in loss of platelet-derived growth factor receptors, exhibit craniofacial and cardiac defects along with a deficit in MMP-2 and MT3-MMP expression [2,69,70].

Moreover, the in vitro migratory capacity of NC-derived craniofacial mesenchymal cells from these mutant mice is reduced [2]. Collectively, these studies suggest MMP-2 and

MT-MMPs have a role in NC morphogenesis and in pharyngeal arch development.

Important insights into the function of these genes can be gained through a close examination of the temporal and spatial expression of MMP-2 and MT3-MMP during NC morphogenesis. To elucidate the role of MMP-2 and MT3-MMP during NC differentiation and migration, I performed a comprehensive study of the temporal and spatial distribution of MT3-MMP mRNA, MMP-2 mRNA and MMP-2 protein during

19 critical stages of early avian NC development. I found that MMP-2 was first expressed

within cardiogenic splanchnic mesoderm before and during the formation of the early

heart tube. While pre-migratory and early migrating NC cells did not synthesize MMP-2,

their interaction with ECM coincided with a redistribution of extracellular MMP-2 that

was deposited by the underlying mesoderm. Soon after populating the pharyngeal arches,

NC cells began synthesizing MMP-2 mRNA. In contrast, NC cells contributing to neural-

related structures (i.e., dorsal root ganglia, sympathetic ganglia, etc.) did not express

MMP-2 mRNA at any stage examined in this study. MT3-MMP mRNA was mainly expressed in mesodermal mesenchyme, mesenchyme of the pharyngeal arches and endocardium but was not detected until stage 13. These findings suggest that MMP-2 plays an important role in NC cell migration and in remodeling of the pharyngeal arches and cardiac tube. MT3-MMP expression suggested that it was only likely involved in these processes once NC cells enter the pharyngeal arches and that activation of secreted proMMP-2 must involve alternative mechanism to MT3-MMP.

20 MATERIALS AND METHODS

In Situ Hybridization

Digoxigenin-labeled antisense and sense riboprobes for chicken MMP-2 and

MT3-MMP (Genbank accession numbers U07775 and U66463 respectively) were generated from a 545-bp BamHl fragment excised from full-length chicken MMP-2 cDNA and a 530-bp EcoRl restriction piece from full-length chicken MT3-MMP cDNA

[71,72], These fragments were subcloned into pBluescript plasmids respectively

(Stratagene, La Jolla, CA), amplified, and sequenced to confirm their identities. After linearizing the plasmid, digoxigenin-labeled sense and antisense riboprobes were generated in the presence of digoxigenin-labeled UTP (Roche Molecular Biochemicals,

Indianapolis, IN) using the T3 and T7 promoters. Hamburger-Hamilton stage 5-25 chick embryos [73] were fixed in 4% paraformaldehyde overnight, rinsed in phosphate- buffered saline (PBS), and prepared for whole mount in situ hybridization. Embryos were hybridized with 2 jug/ml digoxigenin-labeled sense or antisense riboprobe at 58°C for 24 hr. The embryos were then washed with 5X SSC/50% formamide, two changes of 2X

SSC/50% formamide, and finally 2X SSC buffer, all at 58°C. Hybridized digoxigenin- labeled riboprobes were detected using alkaline phosphatase-conjugated sheep digoxigenin antibody (Roche Molecular Biochemicals) and the NBT/BCIP phosphate substrate that produced a bluish/purple insoluble reaction product [74], Whole embryos were refixed in 4% paraformaldehyde, examined, and photographed. Embryos were then embedded in freezing compound and sectioned on a cryostat for further examination.

Hybridization by sense MMP-2 and MT3-MMP riboprobes were not detected in embryos

21 at any stage.

Immunolocalization of MMP-2

Embryos were fixed for 15-30 min in 4% paraformaldehyde in PBS while on ice, washed in PBS, embedded in freezing compound, and sectioned on a cryostat. Sections were equilibrated in PBS, incubated in PBS containing 5% normal goat serum (pH 7.2-

7.4) for 30 mm and then incubated for one hour in rabbit anti-chicken MMP-2 IgG (5-10 pg/ml) in PBS/5% goat serum at room temperature. This antibody was generated against purified recombinant chicken MMP-2, affinity purified, and characterized as described by

Hahn-Dantona and Quigley [75], This antibody binds both proMMP-2 and active MMP-

2. After incubation in this antibody, sections were washed with PBS/0.1% Tween-20 and then incubated in goat anti-rabbit IgG-FITC (4 (tg/ml PBS-5% normal goat serum;

Jackson ImmunoResearch, West Grove, PA) for 1 hr. After washing with PBS-0.1%

Tween-20, sections were coverslipped using Vectashield mounting medium (Vector

Laboratories, Burlingame, CA). Immunostained sections were examined using an epifluorescent-equipped Nikon Labophot-2 microscope and images captured using an digital-cooled SPOT CCD camera (Diagnostic Instruments, Sterling Heights, MI) interfaced to a PowerPC Macintosh computer. Controls included substituting the primary antibody with an equivalent amount of nonimmune rabbit IgG or omission of the primary antibody. No immunostaining was detected in any of the antibody controls. In some cases, sections were also subjected to immunostaining with HNK-1 monoclonal antibody after MMP-2 immunostaining to identify early migrating NC cells [76]. This antibody

22 was procured from conditioned medium of cultured HNK-1 hybridoma cells obtained from American Type Culture Collection (ATCC TIB 200; Rockville, MD).

Zvmo graph v

For zymography, chicken embryos at stage 9/10 and pharyngeal arches containing migrating CNC cells from stage 13/14 embryos were collected and lysed in 0.1 % Triton

X-100. Lysates were centrifuged to remove cellular and ECM debris and the protein content in the supernatant was quantitated using a protein assay (QuantiGold, Diversified

Biotech, Boston, MA). Forty /xg of protein samples were placed in non-denaturing loading buffer and size-fractionated on a gelatin zymogram SDS-PAGE Ready Gel

(BioRad, Hercules, CA). The gel was washed in 2.5% Triton X-100 to remove the SDS, washed three times with water, then incubated for 2 days at 37°C in a 50 mM Tris-HCl buffer containing 10 mM calcium chloride and 0.02% thimerosal, pH 7.4, to permit protease degradation of the gelatin substrate. The gel was subsequently fixed with 50% methanol and 10% acetic acid containing 0.25% Coomassie blue. The gel was then destained with 10% 2-propanol and 10% acetic acid solution. Gelatinase activity appeared as clear bands within the stained gels. Identification of the 72-kDa and 62 kDa forms of MMP-2 in the lysates was determined by comparing the migratory position of the bands obtained using purified chicken MMP-2. The relative degree of gelatinase activity in the zymograms was measured by using a gel documentation system (BioRad,

Hercules, CA).

23 RESULTS

MMP-2 In Situ Hybridization

Digoxigenin-labeled chicken MMP-2 riboprobes were used to examine the spatial and temporal expression of MMP-2 mRNA during NC morphogenesis. MMP-2 mRNA transcripts were first detected at stage 7 in the precardiac mesoderm as the intraembryonic coelom began to form (Fig. 2-1 A). By stage 8, the intensity of MMP-2 mRNA expression was highest in cardiogenic splanchnic mesoderm but transcripts were also detected in the somatic mesoderm and the developing cranial paraxial mesoderm

(Fig. 2-1B). In stage 9 and 10 embryos, the intensity of the hybridization signal increased in the mesoderm especially in the splanchnic mesoderm lining the coelcmic cavity between the developing rostral bulbus cordis and anterior intestinal portal (Fig. 2-1C-E).

MMP-2 transcripts were also detected within developing sclerotome of the rostralmost somites and progressed caudally as new somites formed in older embryos. MMP-2 mRNA was not detected within the ectoderm, endoderm, or in the neural folds or established neural tube; although by stage 12, low levels of MMP-2 mRNA were detected in the ventral floor of the neural tube beginning at the level of the first pharyngeal pouch and extending in the caudal direction (Fig. 2-1H, I, L).

MMP-2 mRNA transcripts were not detected in NC precursor cells within the ectoderm or in segregating and emigrating NC cells. In embryos stages 9+-l 2, early migrating cranial and CNC cells did not express MMP-2 mRNA but were in close proximity to MMP-2 mRNA-expressing cranial mesoderm (Fig. 2-1E-G). By stage 14, all mesenchymal cells within the pharyngeal arches and cranial mesenchyme expressed

24 MMP-2 mRNA, many of which were HNK-1 positive (Fig. 2-11, J). Transcripts were more prevalent in mesenchymal cells in the outer circumferential region of the pharyngeal arches (Fig. 2-1H, I). MMP-2 mRNA was conspicuously absent in NC- containing cranial nerves, dorsal root ganglia, and sympathetic ganglia at all stages examined (Fig. 2-1K-M). Elsewhere in the embryo, the hybridization signal was found in the sclerotome and dermatome and in the endothelium and mesenchyme surrounding the dorsal aortae, and aortic arches (Figs. 2-1H). MMP-2 transcripts were never detected within the cells of the endoderm, ectoderm, myotome, and (Fig. 2-1H).

MT3-MMP In Situ Hybridization

Before stage 13, no MT3-MMP mRNA was detected in chicken embryos using digoxigenin-labeled chicken MT3-MMP riboprobes. After stage 13, MT3-MMP mRNA was detected in paraxial mesoderm, in mesenchyme of the pharyngeal arches, endothelium of great vessels, and in the endocardium and endocardial-derived mesenchyme of the heart (Fig. 2-1N). No mRNA was ever detected in the neural tube, ectoderm, endoderm and myocardium (data not shown). The expression pattern of MT3-

MMP in embryos older than stage 13 was similar to that of the MMP-2. Hybridization of

MT3-MMP antisense riboprobes gradually increased in intensity after stage 13. No hybridization was detected using MT3-MMP sense riboprobes (data not shown).

Zymograms

To confirm the presence of MMP-2, gelatinase activity was assessed in solubilized samples of stage 9/10 chicken embryos and stage 13/14 embryonic

25 pharyngeal arch segments. In stage 9/10 embryos, gelatinase activity at 72 kDa and 62 kDa corresponding to known molecular weights of chicken proMMP-2 and active MMP-

2 w'as detected (Fig. 2-2A). A similar pattern was found using stage 13/14 pharyngeal arches (Fig. 2-2B). No difference in the relative amounts of the 72 kDa or 62 kDa gelatinase was found between younger and older embryos. Including 5 mM EDTA in the incubation buffer blocked all gelatinase activity confirming the lytic bands were generated by metalloproteinases (data not shown).

MMP-2 Immunolocalization

In general, MMP-2 immunostaining paralleled the distribution of MMP-2 mRNA expression where much of the immunostaining was in the form of extracellular particles or associated with cell surfaces. In stage 8 embryos, MMP-2 protein was particularly abundant within the basement membrane underlying the ectoderm, neural folds, notochord, and in the ECM between the cardiac splanchnic mesoderm and foregut (Fig.

2-3A,H). MMP-2 protein was also localized within the cranial paraxial mesoderm, somites, and somatic mesoderm. MMP-2 was not detected within the ectoderm, neural folds, or endoderm. By stage 9 to 9+, mesencephalic NC cells were recognizable as a wedge of cells segregating themselves from the ectoderm during closure (Fig.

2-3B,C). Disruption of the MMP-2 immunostaining in ectodermal/neuroectodermal basement membrane was observed at this site. MMP-2 immunostaining outlined the forming NC cell population and as they emigrated, the NC cells encountered MMP-2 protein localized within the ectodermal basement membrane and underlying mesoderm.

As the NC cells migrated, MMP-2 immunostaining was rearranged at the leading edge of

26 the NC population, became affiliated with trailing NC cells, and appeared to decrease as more NC cells traversed the basement membrane (Fig. 2-3D,E). A similar pattern of immunostaining was observed during subsequent NC cell formation and migration at other cranial axial levels.

In embryos at stage 14-18, MMP-2 protein was found throughout the pharyngeal arch mesenchyme, but immunostaining was more intense in the outer circumference of the pharyngeal arches (Fig. 2-3F,G). More intense immunostaining was evident in the mesenchyme between the aortic sac and endoderm of pharyngeal arches III and IV.

MMP-2 immunostaining also delineated the dorsal aortae (Fig. 2-3F) and developing blood vessels and was found in developing sclerotome and somatic mesoderm of the lateral body wall. MMP-2 protein was not found within endodermal-derived primordia of the liver, lung, and thyroid gland or within ectodermal derivatives including the neural tube, optic and otic vesicles, developing lens, cranial nerves, and sympathetic and dorsal root ganglia. No immunostaining was seen in the control (Fig. 2-3H).

27 DISCUSSION

MMP-2 and NC Cells

Our results showed that emerging and early migrating NC cells did not synthesize

MMP-2 mRNA but that they could interact with extracellular MMP-2 protein synthesized by the mesoderm. Immunostaining of the ectodermal basement membrane was disrupted at the site at which NC cells emerged from the neural tube, paralleling the disruption of fibronectin, laminin, and type IV collagen in the basement membrane during trunk NC emigration [77], MMP-2 degrades many ECM components including type IV and type V collagen, laminin-5, elastin, fibronectin, and proteoglycans [14,78,79], Therefore, once activated, MMP-2 could be responsible for disrupting the distribution of basement membrane components adjacent to the site of NC emigration. As cranial NC cells moved beneath the ectoderm, MMP-2 immunostaining of the ectodermal basement membrane was disrupted near the NC front and MMP-2 became associated with the surfaces of NC cells, and diminished in intensity as trailing NC cells traversed the basement membrane.

The NC-associated MMP-2 was probably secreted by mesoderm and recruited by NC cells, since we did not detect MMP-2 mRNA in NC cells at this stage. This MMP-2 may facilitate the migration and invasion of NC cells by degrading ECM and disrupting cell/ECM interactions. MMP-2 mediated ECM degradation may also be responsible for the loss of basement membrane-associated ECM at the migratory front of migrating NC cells [80], Interestingly, the migration pattern and developmental fate of late migrating mesencephalic NC cells is influenced by environmental changes that depend on earlier migration of NC cells at this level [81]. Therefore, modification of the MMP-2

28 distribution and/or ECM proteolytic degradation by pioneering NC cells may reflect these environmental changes or be responsible in part for directing the migration pattern and developmental fate of trailing NC cells.

In embryos stage 13 and older, MMP-2 mRNA expression increased within the cranial mesoderm and in NC-derived mesenchyme found in the pharyngeal arches, maxillary and mandibular process, outflow tract, and surrounding the optic and otic vesicles. However, NC cells forming dorsal root ganglia, sympathetic ganglia, or cranial nerve ganglia did not express MMP-2 mRNA. This implies MMP-2 expression by NC cells may be regulated by specific tissue -tissue interactions or may reflect NC lineage restrictions specified before or at the time of their emigration. This also suggests that the ability to express MMP-2 may be a characteristic of NC lineages forming non-neural tissue-related cell types. However, NC cells forming neural tissue-related cell types could still use MMP-2 derived from other sources.

Recently, transgenic MMP-2 and MMP-9 null mice have been generated [44,50],

These null mice are bom with normal appearing phenotypes, are viable, and fertile.

However, MMP-9 null mice exhibit delayed long bone osteogenesis. Aberrant apoptosis, vascularization, and ossification eventually compensate for the delay in osteogenesis so that by two months postnatal age, MMP-9 null mice exhibit normal appearing bones [44].

Therefore, in gene-null animals, quite normal phenotypes can be obtained by enlisting different and alternative developmental mechanisms. Because NC and cardiac morphogenesis have not been examined in MMP-2 and MMP-9 null mice, it is still unclear whether the developmental course for NC morphogenesis or great vessel and cardiac organogenesis is identical to wild-type embryos. Moreover, determining the

29 precise role of specific extracellular and cell-surface proteases in transgenic animals is difficult given the wide overlap in substrate specificities between the metalloproteinases.

Further study of the development of other NC-containing organs in these mice may yield important clues as to the developmental role of MMP-2.

MT3-MMP and NC cells

MT3-MMP mRNA was not detected in chicken embryos until stage 13. After

stage 13, detectable levels of MT3-MMP mRNA were found in the paraxial mesoderm, pharyngeal arch mesenchyme, endothelium of great vessels, endocardium and endocardial-derived cushion cells of the heart. These data are in accordance with what has been reported by Alexander et. al. (1997) [40], In their study using RT-PCR, they detect only very small levels of MT3-MMP mRNA in the stage 12 quail embryonic hearts, but levels of MT3-MMP mRNA increase significantly after stage 12. The difference in sensitivity between in situ hybridization and RT-PCR or slight species differences could account for the discrepancy between my findings and theirs.

Nevertheless, the increase in MT3-MMP mRNA expression after stage 13 observed by in situ hybridization closely matches what is reported for quail embryos.

MT3-MMP was not detected in chicken embryo before stage 13, suggesting this

MT-MMP is not likely involved in the formation and early migration of CNC cells. Yet, zymography showed that active MMP-2 was present in stage 9/10 chicken embryos. This suggests that another MT-MMP may be expressed in these early chicken embryos since proMMP-2 is primarily activated by MT-MMPs in vivo [18], To date at least 6 types of

MT-MMPs have been cloned and characterized [67,82,83] and many have been shown to

30 directly digest interstitial collagens, gelatin, and other ECM macromolecules in addition to activating proMMP-2 [28,84,85], A likely candidate for activation of proMMP-2. in chick embryo is MT1-MMP. MT1-MMP has been shown to be localized at the surface of invadopodia in invasive cells [86], Moreover, in transgenic mice MT1-MMP null mutants have craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity [48], Cloning and characterization of this molecule in chicken model will be needed in order to determine if NC cells express this molecule and if it is essential for proMMP-2 activation in these early embryos.

In embryos older than stage 13, MT3-MMP was expressed in the paraxial mesoderm, pharyngeal arch mesenchyme, and endothelium of great vessels, largely paralleling MMP-2 expression. In pharyngeal arches, some NC cells will form the tunica media, differentiating into smooth muscle cells and fibroblasts ensheathing the great vessels [60]. Interestingly, MT3-MMP has been reported as having a role in blood vessel remodeling through direct proteolysis of ECM and by activation of proMMP-2 [87,88],

Therefore, MT3-MMP expressed in pharyngeal arches could have a role in normal great vessel development. In addition, the Patch mutant mouse further provides some insights regarding the functional role of MT3-MMP [2], MT3-MMP mRNA expression is reduced by 95% in patch mutants based on semiquantative RT-PCR with a concomitant reduction in migratory ability of pharyngeal mesenchyme (mostly NC-derived) in vitro. Migration of these cells can be rescued by adding platelet-derived growth factor into the culture media which is accompanied with a significant elevation of active MMP-2. These data suggest MT3-MMP activates proMMP-2 and mediates NC cell migration during pharyngeal arch development and remodeling. MT3-MMP in situ hybridization results

31 shown in this study supports this postulate.

In summary, we showed that (1) the temporal and spatial distribution of MMP-2 mRNA and protein was consistent with MMP-2 having an integral role in mediating

CNC morphogenesis, (2) MMP-2 was primarily expressed within mesenchyme of mesodermal origin and in older pharyngeal CNC populations, (3) NC cells encountered and altered the distribution of MMP-2 associated with cranial ectodermal basement membrane, and (4) MT3-MMP is not likely involved in early CNC emigration or migration but may play a role in subsequent pharyngeal arch and cardiac development.

MMP-2 functional activity reflects a culmination of MMP-2 synthesis and secretion, receptor binding and activation, and interactions with local inhibitors. Hence, future studies must include a careful examination of the expression of these molecules as well.

However, performing experiments that directly test whether MMP activity is required for normal NC and cardiac development would seem a reasonable initial step.

32 Figure 2-1. MMP-2 and MT3-MMP mRNA expression during early neural crest morphogenesis. (A) MMP-2 mRNA expression in a stage 7 embryo showing earliest detectable MMP-2 mRNA in precardiac lateral plate mesoderm (asterisk). (B) MMP-2 mRNA in lateral plate mesoderm of a stage 8 embryo. Most intense hybridization signal was found in the splanchnic mesoderm. Signal was also detected in the paraxial mesoderm. (C) Whole mount MMP-2 in situ hybridization of a stage 9+ embryo, ventral view. Cells lining the coleomic cavity, developing myocardium, and cranial paraxial mesoderm expressed MMP-2 mRNA. (D) MMP-2 expression in bulbus cordis region of stage 10+ embryo. (E) MMP-2 mRNA expression in a stage 10 embryo. MMP-2 transcripts were present within paraxial mesoderm (pm) but not in migrating NC cells

(asterisk) or neural tube. (F) MMP-2 expression during stage 10 at level of anterior rhombencephalic level, asterisk, NC cells. (G) HNK-1 immunostaining of the section shown in F. HNK-1 positive NC cells did not express MMP-2 mRNA but were surrounded by MMP-2-expressing mesoderm. (H) MMP-2 expression in a stage 17 embryo at the level of the pharyngeal arches (arches I, II, III, and IV). MMP-2 expression was prominent in pharyngeal arch mesenchyme, particularly in the outer circumferential area beneath the ectoderm. (I) MMP-2 mRNA was expressed in all pharyngeal arch mesenchyme at stage 14. (J) HNK-1 immunostaining of the section shown in I. HNK-1 antibody bound to the otic pits and a portion of the pharyngeal arch mesenchyme. (K)

MMP-2 mRNA expression was not detected in NC-containing cranial nerves. This sample is from a stage 21 embryo. (M) HNK-1 immunostaining of the section shown in

L. HNK-1 antibody bound to the NC-derived dorsal root oi sympathetic ganglia. (N)

Whole mount MT3-MMP in situ hybridization of a stage 15 embryo, side view.

33 Endocardium of heart, mesenchyme in pharyngeal arches, and paraxial mesoderm expressed MT3-MMP RNA. The message was not detected in myocardium and ectoderm. Abbreviations: cn, fifth cranial nerve; d, dorsal aorta; drg, dorsal root ganglia; ed, endocardium; en, endoderm; et, ectoderm; ic, intraembryonic coelom; m, myocardium; my, myotome; n, neural folds or neural tube; ot, otic pit; pm, paraxial mesoderm; sp, splanchnic mesoderm. Scale bar = 50 pm in A, E-G; 52 pm in D; 100 pmin B, I, J, L, M; 140 pm in K; 150 pm in H; and 830 pm in C and N.

34 Figure 2-1

35 Figure 2-2. Image of a gelatinase zymogram prepared using equal amounts of protein from tissue lysates of whole mount embryos of stage 9/10 (A) and tissue lysates of pharyngeal arch from stage 13/14 embryos (B). Lytic bands (white bands) were found at approximately 72 kDa and 62 kDa. No change between the relative proportion of 72 kDa and 62 kDa gelatinase activity (arrowheads) was detected between stages 9/10 and 13/14 as determined by scanning densitometry.

36

Figure 2-3. MMP-2 immunostaining. (A) MMP-2 immunostaining in a stage 8 embryo.

Immunostaining was found within the extracellular matrix (ECM) of the mesoderm and in basement membranes underlying the ectoderm, neural tube, and endoderm. (B) MMP-

2 immunostaining outlined the basement membrane of the ectoderm and neural tube as well as the wedge of forming NC cells. (C) MMP-2 immunostaining in another stage 9+ embryo (NC cells, asterisks). (D) MMP-2 immunostaining in early migrating cardiac NC cells of a stage 11- embryo. Immunostaining along the ectodermal basement membrane

(arrowheads) was disrupted at the leading front of migrating NC cells. (E) HNK-1 immunostaining of the same section shown in D labeling the migrating NC cells. (F)

MMP-2 immunostaining at the third pharyngeal arch level of a stage 15 embryo. MMP-2 immunostaining was prevalent in the pharyngeal arch mesenchyme (asterisk) and was localized in the cardiac ECM. (G) MMP-2 immunostaining within the mandible and second pharyngeal arch of a stage 17 embryo. (H) MMP-2 immunostaining was prevalent in ECM between the cardiac splanchnic mesoderm and endoderm (asterisks) at stage 8.

(I) A control from a stage 8 embryo using a nonimmune rabbit IgG as the primary antibody. No immunostaining was found. Abbreviations: as, aortic sac; d, dorsal aorta; en, endoderm; et, ectoderm; m, myocardium; n, neural folds or neural tube. Scale bar =

50 (am in C-F; 100 pm in A, B, G, and FI.

38 G • *• ■ V-;. * ■*» ; II ?

%

% - . v Vh. 1 : ■

Figure 2-3

n CHAPTER THREE

A SYNTHETIC MMP INHIBITOR DECREASES EARLY

CARDIAC NEURAL CREST MIGRATION IN CHICKEN

EMBRYOS

40 INTRODUCTION

Neural crest (NC) cells are an embryonic migratory cell population that develops at the border between the neural plate and the ectoderm. As the neural plate closes to form the neural tube, NC cells emigrate from the epithelium in a rostro-caudal wave and migrate throughout the embryo. NC cells form a wide range of cell types important in the formation of the skin, craniofacial structures, cardiovascular system, endocrine organs, and much of the peripheral [89], NC cells arising between the mid-otic and caudal end of 3rd somite axial level (dubbed “cardiac NC cells), migrate beneath the ectoderm into the pharyngeal arches 3, 4, and 6. Once there, a subset of these cells invade the outflow tract of the heart and form two spiraling ridges that participate in the septation of the outflow tract into the aorta and pulmonary artery [62,90], Ablation studies show that severe cardiac defects such as persistent truncus arteriosus, double-outlet right ventricle, and tetralogy of Fallot, occur without these cardiac NC (CNC) cells [54] thereby mirroring several human congenital defects like DiGeorge syndrome and velocardiofacial syndrome.

Although NC cells have been studied for more than 130 years and much is known about their migration pathways and their derivatives [89,91], the mechanisms enabling and directing NC cell migration are still elusive due to the complexity of the cellular and molecular interactions involved. To date, two interplaying mechanisms are thought to be central for the guidance of the migrating NC cells: one involves the release of signaling molecules acting through their cognate protein kinase/phosphatase-type receptors, and the other is through the interactions of the cells with their surrounding extracellular matrix (ECM)

[92], The former suggests that different NC subpopulations begin to express specific receptor

41 tyrosine kinases after they leave the neural tube in response to presently unknown intrinsic or extrinsic cues, thereby allowing them to respond to differentially-localized growth factor ligands in the embryo. The latter, emphasizes the critical role played by intracellular signals transduced through integrin/ECM interactions [91,92], The expression of various integrins by

NC cells and the differential distribution of migration-permissive and non-permissive ECM molecules have been well documented and favor the role of the integrin/ECM model for directing NC cell migration [92], Researchers have focused on synthesis, secretion and assembly of ECM components encountered by migrating NC cells but have paid little attention to the turnover or remodeling of ECM components that would also be necessary for

NC cell migration.

Matrix metalloproteinases (MMPs) are a family of more than 20 zinc-containing enzymes that play important roles in the degradation and turnover of ECM. Based on their structures and substrate preferences, MMPs can be roughly divided into four subfamilies: collagenases, gelatinases, stromelysins, and membrane-type MMPs (MT-

MMPs) [67]. Collagenases and gelatinases are secreted as zymogens (proMMPs) that are activated extracellularly by serine-proteases like plasmin, membrane-intercalated MT-

MMPs, or by autoactivation. In contrast, stromelysin and MT-MMPs are activated intracellularly by enzymes of furin family [8]. MMPs degrade almost all known ECM components including interstitial collagens, proteoglycans, elastin, laminin, fibronectin, and others. Studies also show that MMPs in different family groups share common substrates. For example, gelatinase A (MMP-2), MT1-MMP, and MMP-1 can all degrade interstitial collagen I [64,84].

42 The involvement of MMPs in cell migration has been widely studied. Many malignant cells synthesize and secrete MMPs or encounter MMP molecules secreted by neighboring stromal cells as they migrate [93,94], Natural or synthetic MMP inhibitors reduce tumor cell migration or metastasis in vitro and in vivo (reviewed by Chambers and

Matrisian) [95]. Like tumor cells, NC cells penetrate through the basement membranes and invade ECM during their emigration and migration. Therefore, NC cells likely employ migratory mechanisms that are similar if not identifical to the ones employed by tumor cells. In Chapter 2, the spatial and temporal distribution pattern of MMP-2 was shown to correlate with CNC migration in chicken embryos. Furthermore, studies in our lab showed that a synthetic MMP inhibitor decreased CNC cell motility in vitro (Brauer, unpublished data). These data suggest that MMP activity may play a role in mediating

NC migration in vivo. In this chapter, I tested the hypothesis that microinjecting a synthetic MMP inhibitor, KB8301, into the CNC migratory pathway of embryos would inhibit CNC cell migration. My data showed that KB8301 decreased the MMP enzymatic activity levels in embryos and significantly decreased the distance CNC cells migrated in vivo. This supports the idea that MMP enzymatic activity is an important mediator of

CNC cell migration in vivo.

43 MATERIALS AND METHODS

MMP Activity Assay

Fertilized White Leghorn chicken eggs were incubated at 38°C in a 60% relative humidity environment until embryos reached Hamburger-Hamilton stage 10 [73].

Following removal of 1 ml of albumin, eggs were windowed to reveal the embryo below.

A 1:20 dilution of India ink in Ringer’s solution was injected beneath the blastoderm in order to better visualize the embryos. The vitelline membrane was tom with a sharp tungsten needle to expose the area between the mid-otic and the 3rd somite level of the embryo. KB8301 (46 or 92 pmoles each side) in DMSO was bilaterally injected beneath the ectoderm between the mid-otic to 3rd somite level using a “Nanoject” automatic oocyte injector (Drummond Scientific Co., Broomall, PA). Eggs were refilled with albumin, sealed with vinyl tape, and reincubated for 6 hr. Controls included the same stage embryos injected with equivalent concentration and volume of DMSO. After 6 hr the embryos were collected, tissue blocks between mid-otic and 3rd somite level were excised, and splanchnic mesoderm and heart were carefully removed by microdissection from the collected tissues. The tissues was homogenized by vortexing in 0.1% Triton X-

100 for 10 min and supernatants were collected after centrifugation at 12,000g for 10 min.

A fluorescent method was used to measure MMP enzymatic activity in the tissue lysates [96]. Briefly, four /d of the tissue lysate supernatant was incubated with 12.5 fx 1 of

0.5 mM synthetic MMP substrate, MoCA-Pro-Leu-Gly-Leu-[3-DNP-2,3-DAP]-Ala-Arg

(American Peptide Co., Sunnyval, CA), in 250 /d of assay buffer (0.1 M Tris-HCl, 0.1 M

44 NaCl, 10 mM CaCl2, and 0.05% Brij-35, pH 7.5) for 20 min at 37°C. After cleavage of the gly-leu bond, this substrate fluorescences at a 390.0 nm wavelength when excited at a wavelength of 326.1 nm. Fluorescence was read on a TD-700 Fluorometer (Turner

Designs, Sunnyvale, CA). The enzymatic activity of the samples was calculated based on a standard curve using known amounts of active human MMP-2. (Alexis Biochemicals,

San Diego, CA). MMP enzymatic activity was normalized to the concentration of DNA in the lysate. DNA was measured using the Hoescht dye H33258/TKO 100 DNA assay method on a minifluorometer (Hoefer Scientific Instruments, San Francisco, CA) [97].

The enzymatic activity assay was repeated three (for 46 pmoles of KB8301) or four times

(for 92 pmoles of KB8301). The ratio of the enzymatic activity ((ill of MMP-2 activity/pg DNA/time) of KB8301 injected to sham controls was compared to the ratio of enzymatic activity in DMSO injected to sham controls using a non-paired t test. The test was considered statistically significant when the p value was less than 0.05.

KB8301 Injection and CNC Migration

KB8301 (46, 92, or 184 pmoles) was microinjected unilaterally into the cell-free space adjacent to the premigratory CNC cells at the 2nd somite level of chicken embryos at stages 10', 10, and 10+. Control embryos received an equivalent volume of the KB8301 vehicle (DMSO). Eggs were sealed and returned to the incubator for 13 hr by which time embryos were at stages 13-14. Embryos were collected, fixed in 4% paraformaldehyde, permeabilized with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1%

SDS, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0) for 3 hr. Embryos were then incubated at

4°C overnight with a 1:3 dilution of hybridoma conditioned culture medium (ATCC,

45 Manasus, MA) containing the HNK-1 IgM antibody that recognizes an epitope on NC cells. The embryos were washed 5 times with PBS-0.1% Tween-20, and then incubated with 6 /xg/ml donkey anti-mouse IgM-TRITC (Jackson ImmunoResearch, West Grove,

PA) at 4°C overnight. The embryos were then washed in PBS-0.1% Tween-20 and refixed in 4% paraformaldehyde, dehydrated, and embedded in JB-4 plastic (EMS, Ft.

Washington, PA). Four-micron-thick serial cross sections were prepared using a Leica

RM2135 microtome (NCI, Minneapolis, MN). Serial sections at the 2nd somite level were stained with 0.5 pg/ml of DAPI (Sigma Chemical Co., St. Louis, MO) to label the nuclei.

Finally, the sections were examined and photographed with epifluorescence-equipped microscopy and a color CCD camera. HNK-1 and DAPI images of the same tissue section were combined using IPLab image analysis software (Scanalytics, Inc, Fairfax,

VA). The distance between the dorsal midline of the neural tube to the leading edge of

CNC cells was measured along the basal surface of the ectoderm. Five serial tissue sections, beginning with the most cranial section containing the 2nd somite, were used for the measurement and migratory distance of the leading CNC cells on each side was averaged separately for each embryo. All measurements were made in the blind. The migratory distance between the injected and noninjected side of KB8301-treated or

DMSO-treated embryos was then compared using a paired t test. The migratory distance in the injected side of KB8301-treated and DMSO-treated embryos was also compared using a non-paired t test. To exclude possible effects of KB8301 on embryo size, the ratio of the migratory distance in the injected side and the noninjected side was compared between the DMSO-treated and KB8301-treated embryos. Finally, a 2-way ANOVA was adopted to analyze the possible effect of KB8301 and DMSO on developmental rate (end

46 stage) of the collected embryos. Tests were considered statistically significant at p values

< 0.05.

47 RESULTS

Effect of KB8301 Injection on MMP Activity

To determine whether microinjection of KB8301 decreased MMP activity in vivo,

MMP enzymatic activity in embryos were measured 6 hr after KB8301 injection.

Bilateral injection of 92 pmoles of KB8301 significantly reduced the level of MMP activity in treated embryos compared to DMSO (carrier)-treated embryos. Bilateral microinjection of 46 pmoles of KB8301 did not significantly decrease MMP activity in treated embryos (Table 3-1).

Effect of KB8301 on Embryonic Development

Injection of DMSO had no noticeable effect on the overall development of embryos. Approximately 95% (90 out of 95 embryos) and 92% (83 out of 90 embryos) of the embryos treated with the concentrations of DMSO used for 46 or 92 pmoles of

KB8301 were alive, were of comparable size, and were morphologically indistinguishable from sham operated embryos (Fig. 3-lA,B). Visual assessment of

HNK-1 immunostaining also suggested that cranial, cardiac, and trunk NC cells emigrated and migrated normally in DMSO-treated embryos. Most of the embryos unilaterally injected with 46 or 92 pmoles of KB8301 at the 2nd somite level also survived (93%, 102 out of 110 embryos treated with 46 pmoles, and 91%, 90 out of 99 embryos treated with 92 pmoles) and had no distinguishable morphological differences compared with their responding controls (Fig. 3-1C). HNK-1 immunostaining showed

48 that cranial and trunk NC cells emigrated and began migrating in embryos injected with

46 or 92 pmoles of KB8301. However, the extent of NC migration in the injected side of embryos treated with 92 pmoles of KB8301 appeared to be decreased compared with the contralateral noninjected side (Fig. 3-1C). Unilateral injection of 184 pmoles of KB8301 was devastating in that approximated 60% of treated embryos (43 out of 72 treated embryos) either died soon after injection or exhibited severe distorted morphologies such as failure of neural tube closure, lesions in the injection spot, and missing cranial structures. The 40% of embryos that survived exhibited a decreased growth rate, reduced somite size on the injected side, and abnormally curved cranial-caudal axis (Fig. 3-ID).

Few HNK-1 positive cranial NC cells and CNC cells were observed in embryos treated with 184 pmoles of KB8301 but the trunk NC cells appeared to emigrate and migrate normally. The was also HNK-1 negative in these embryos (Fig. 3-ID).

To determine whether KB8301 injection caused cell death, serial sections of embryos injected with different concentrations of KB8301 or DMSO were stained with the DNA-binding fluorescence dye, DAPI. There were no observable difference of pyknosis, karyorrhexis, or karyolysis in CNC cells (identified by HNK-1 immunostaining), somitic cells, paraxial mesodermal cells, or ectodermal cells in the embryos injected with DMSO, 46 pmoles of KB8301, or 92 pmoles of KB8301 (Fig. 3-

2A,B). However, CNC cells, neural tube cells, and paraxial mesodermal cells in embryos injected with 184 pmoles of KB8301 exhibited signs of nuclear fragmentation, suggesting large-scale cell death with KB8301 at this concentration (Fig. 3-2C). Therefore, only 46 and 92 pmoles of KB8301 were used in subsequent experiments.

49 To determine if KB8301 altered the developmental rate of embryos, I compared the developmental stage of embryos 13 hr after treatment with DMSO or KB8301. Based on Hamburger and Hamilton staging, the developmental rate of embryos treated with 46 or 92 pmoles of KB8301 was not significantly different compared with that of DMSO controls (Table 3-2). However, there was a tendency for the development of KB8301- treated embryos to be retarded compared to the DMSO controls, particular by evidence in embryos injected at stage 10'. Embryos injected with 184 pmoles of KB8301 were not analyzed due to high mortality and abnormalies.

Effect of KB8301 on CNC Migratory Distance

To determine if KB8301 inhibited CNC cell migration, the distance CNC cells migrated from their origin (dorsal apex of the neural tube) was measured in cross- sections of injected embryos at the level of the 2nd somite (Fig. 3-3A,B). CNC cell migratory distance in chicken embryos injected with either DMSO or 46 pmoles of

KB8301 at stages 10', 10, or 10+was not significantly different between the injected side and noninjected side. In addition, there was no difference in the average distance CNC cells migrated on the injected side of DMSO-treated embryos versus the distance in

KB8301-treated embryos (Fig. 3-4).

When embryos were injected with 92 pmoles of KB8301 at stages 10', 10, or 10+, the distance CNC cells migrated in the injected side was significantly decreased compared to that in the noninjected side. DMSO-injected embryos exhibited no measurable difference in migration between the injected and noninjected side (Fig. 3-5).

50 Moreover, the average migratory distance of CNC cells in the KB8301-injected side was significantly reduced compared to that of the DMSO-injected side. (Fig. 3-5).

Although KB8301 injection did not affect the rate of early embryonic development based on staging, the difference in migratory distance between DMSO- treated and KB8301-treated embryos could be attributable to changes in embryo size. To evaluate this possibility, the ratio of the CNC migratory distance between the KB8301- injected side and noninjected side was compared to the ratio found in the DMSO-treated embryos. This normalizes for any differences in embryo size when assessing CNC migratory distance. Again, KB8301-treated embryos showed a significant reduction in the average distance CNC cells migrated compared to DMSO-treated embryos (Table 3-

3).

As another means to assess the possibility that effects of KB8301 on CNC cell migratory distance might be attributable to differences in embryo size, the cross-sectional area of the neural tube in both DMSO-treated and KB8301-treated embryos was measured at the 2nd somite level and compared. The neural tube area was not significantly different between DMSO-treated and KB8301-treated embryos (Fig. 3-6).

Although embryos injected at a particular stage were subsequently reincubated for

13 hr, the developmental stage at the end of this period varied. To determine if the effects of 92 pmoles of KB8301 on migratory distance might be attributable to differences in their subsequent developmental rate, a two-way ANOVA was performed with the end stage being the independent variable. Regardless of the end stage, the migratory distance of CNC cells in embryos treated with 92 pmoles of KB8301 was always significantly less than that in DMSO-treated stage 10 embryos (p < 0.001) (Table 3-4).

51 DISCUSSION

My previous work characterizing the expression pattern of MMP-2 and MT3-

MMP during early CNC and cardiac morphogenesis suggested MMP-2 is correlated with

NC cell migration (described in Chapter 2). In this study, I showed that injecting a synthetic MMP inhibitor into embryos decreased MMP activity as well as CNC cell migration in vivo. This is the first known evidence showing MMP enzymatic activity is an important mediator of early CNC cell migration in vivo.

KB8301 and MMP Enzymatic Activity

Due to their potential pharmaceutical uses, many MMP synthetic inhibitors have been developed based on the chelation of zinc through the use of thiols, carboxyls, phosphoruses, and hydroxamates [67]. Hydroxamates have been widely used to study functional roles of MMPs in normal cell migration and malignant cell metastasis because of their stability and low IC50 values. Due to the structural conservation of the catalytic domain among MMPs, hydroxamates like KB8301, BB-94, GM6001, Ro31-9790, and

BB2516 are all nonselective inhibitors. Hydroxamates have also been used to examine

the relationship between MMP activity and cell migration in vitro and in vivo. For

example, Chin and Werb (1997) blocked MMP activity as well as cell migration in

explanted embryonic mandibles using a synthetic MMP inhibitor [3], Song et. al. (2000)

microinjected BB-94 into quail embryonic heart inhibiting MMP-2 activity and cushion

cell migration [41], These results showed the effectiveness of these synthetic inhibitors.

52 KB8301 strongly inhibits MMP-1, MMP-2, MMP-3, MMP-9, ar.d other MMPs with a low IC50 (0.3 - 0.6 nM) [98], It has been used to inhibit MMP-2 activity in explanted mouse pharyngeal arches [2], sheddase activity in TNF-induced shedding of

TNF receptors [99], and in metalloproteinase-mediated release of Fas ligand [100], In this study, bilateral microinjection of KJB8301 (92 pmoles each side) into stage 10 chicken embryos significantly decreased the enzymatic activity of MMPs. When KB8301 was added to samples of tissue lysates of untreated embryos, MMP activity was also decreased (data not shown). In addition, adding KB8301 to the incubation buffer of gelatin zymograms of chicken embryonic pharyngeal arch or heart lysates significantly reduces MMP-2 enzymatic activity (Brauer, unpublished data). These results establish that KB8301 inhibits chicken MMP activity in vitro as well as in vivo.

MMP Activity and Embryonic Development

Studies suggest that MMPs are important for embryonic development. For example, several MMPs and TIMPs are expressed in maternal decidua and its stroma during mouse embryo implantation and a synthetic MMP inhibitor delivered intraperitoneally reduces decidual length and overall size of the embryo [101], Several

MMPs and TIMPs are expressed in embryonic day 13-15 mouse mandibles and tongue muscles and a synthetic MMP inhibitor dramatically alters the morphogenesis of cultured explanted mandibles [3], Mouse strains deficient in several individual MMPs (MMP-2,

MMP-3, MMP-7, MMP-9, MMP-12, MT1-MMP) have been developed. MMP-2 null mutants show decreased growth rate and short limbs while MT1-MMP deficiency gives nse to a set of severe, generalized connective tissue abnormalities resulting in dwarfism

53 and early death [43-49], These studies support the idea that at least some of the MMPs are important for embryonic morphogenesis and that synthetic MMP inhibitors are able to alter embryonic development.

In this study, injection of 46 or 92 pmoles of KB8301 had no measurable affect on overall embryonic morphogenesis or the rate of embryonic development even though

MMP activity was decreased by 30% in embryos treated with 92 pmoles of KB8301.

However, embryos injected with 92 pmoles of KB83Q1 tended to be smaller than DMSO controls although there was no significant difference between them. These visually

“normal” embryos might harbor minor developmental abnormalities that may only be apparent with closer examination or after reaching older stages. In fact when measured, embryos treated with 92 pmoles of KB8301 showed decreased CNC migration distance on the injected side. Raising the dose of KB8301 to 184 pmoles induced high mortality or severe defects in treated embryos and HNK-1 staining labeled few CNC cells, suggesting

CNC cells failed to form or emigrate from neural tube. Therefore, maintaining particular levels of MMP activity in vivo appears important for embryonic morphogenesis.

KB8301 and CNC Cell Migration

Injecting KB8301 into embryos just prior to CNC emigration decreased the average distance CNC migrated by approximately 15% on the injected side compared to the noninjected side. This decrease was not attributable to changes in developmental rate or embryo size suggesting that KB8301 reduced the local migratory capacity of CNC cells rather than having a global effect on embryos. KB8301 also decreased MMP activity in embryos suggesting that the action of KB8301 on CNC migration was

54 mediated through its ability to inhibit MMP activity. While the degree of enzymatic inhibition (-30%) was greater than the corresponding decrease in CNC migratory distance (-15%), this difference could be the result of decreasing in vivo local concentrations of KB8301 due to molecular diffusion, turnover, and/or the increasing size of the embryo. In addition, the loss of MMP activity may be compensated for by an increase in MMP synthesis, increase in activation of proMMPs, or decrease in synthesis of MMP inhibitors. Moreover, there may be an upregulation of other proteinases, e.g., urokinase and plasmin which have been shown to be involved in NC migration

[102,103], that are unaffected by KB8301. Regardless, KB8301 inhibited MMP activity and reduced the distance CNC migrated from the neural tube providing evidence that

MMP activity is an important mediator of CNC migration in vivo.

The decrease in migratory distance observed in KB8301-treated CNC cells could result from a decrease in CNC cell motility, ECM invasiveness, or decrease in CNC emigration from the neural tube. KB8301 decreases motility and increases cell surface area of CNC cells in vitro (Brauer, unpublished observations). Robbins et al. (1999) showed that KB8301 decreased the migration of NC-derived pharyngeal arch mesenchyme in vitro. In addition, other hydroxamate MMP inhibitors decrease the motility of tumor cells and normal cells in vitro as well with corresponding decreases in

MMP activity. For example, marimastat, BB-94, and batimastat markedly reduced the motility of glioma cells, human hepatocellular carcinoma cells, and keratinocytes respectively [104] [105,106], These observations suggest that reduction of in vivo CNC migratory distance in KB8301 injected sites was due in part to a decreased CNC cell motility.

55 KB8301 could also decrease migratory distance by reducing ECM degradation, thereby reducing invasiveness. Hydroxamate MMP inhibitors similar to KB8301 reduce degradation of ECM and corresponding cellular invasion. For example, GI12947 inhibits

MMP-mediated collagen cleavage and reduces ovarian cancer cell invasiveness in vitro

[107] while BB3103 decreases the level of degradation of MMP-dependent fibrillar type I collagen and angiogenesis in vivo [108]. Evidence suggests that NC cells also degrade

ECM as they migrate [80,109]. As described in the previous chapter, the extracellular distribution of MMP-2 within ECM was disrupted at migratory CNC front and MMP-2 immunostaining appeared reduced within the trailing CNC migratory population. This further supports the idea that CNC cells degrade ECM as they migrate. While not specifically examined in this study, KB8301 could restrict CNC migration by reducing iocal MMP-dependent ECM degradation and hence CNC invasiveness.

To generate CNC cells, precursor cells must undergo an epithelial-mesenchymal transition necessitating major changes in cell-cell adhesion and/or removal of the underlying basement membrane. Inhibitors of MMPs can block similar epithelial- mesenchymal cell transition occurring during tumor metastasis while reducing endogenous MMP inhibitor (e.g., TIMPs) levels can increase metastasis [23,110-114].

Studies also suggest a direct relationship between N-CAM and E-cadherin expression and

MMP activity. Overexpressing stromelysin-1 in mammary epithelial cells increases degradation of E-cadherin, disrupts cell-cell adhesion, and leads to an epithelial- mesenchymal cell transition of these cells [115]. Increasing N-CAM or E-cadherin expression reduces MMP activity and metastasis [116,117]. Therefore, KB8301 may

56 decrease NC migratory distance by inhibiting MMP activity needed for the epithelial- mesenchymal transition process responsible for generating NC cells.

The basement membrane is discontinuous at sites where NC cells emigrate [77],

While the mechanism for this discontinuity is unclear, it likely involves MMPs (e.g.,

MMP-2) that can degrade type IV collagen. In the previous chapter, MMP-2 immunostaining was found in this basement membrane suggesting that local MMP-2 activity could contribute to the discontinuity of the basement membrane barrier.

Therefore, KB8301 could delay the emigration of NC cells by preventing the loss of basement membrane integrity. In my pilot studies using cultured embryo slices, when

KB8301 was added to the medium, CNC cells accumulated at the dorsal neural tube giving the appearance that they were unable to escape the epithelium (unpublished observation). This was also observed in some embryos injected with high amounts of

KB8301. A similar phenomenon was reported by Bronner-Fraser after injecting embryos with N-CAM antibodies [31]. These observations suggest that MMP activity plays an important role in the segregation of CNC precursors and their subsequent emigration from the neural tube.

In addition to inhibiting MMP activity, KB8301 can inhibit some non-matrix metalloproteinases like ADAMs (A Disintegrin And Metalloproteinase domain).

ADAMs are involved in a number of biological processes, including fertilization, neural

specification, cell migration, and muscle differentiation [118]. Two of the ADAMs

(ADAM-13 and xmdc-11) are expressed by Xenopus cranial NC cells and the catalytic

activity of ADAM-13 is required for cranial NC migration [119-122], Therefore, the effect of KB8301 on NC migration could be attributed to the altered function of ADAM-

57 13. However, this will require further examination as we do not yet know the pattern of

ADAM expression in chicken embryos and whether KB8301 inhibits ADAM-13 enzymatic activity.

In summary, microinjection of the MMP synthetic inhibitor, KB8301, into the

CNC cell migratory pathway decreased both MMP activity and CNC cell migration.

These results suggest that enzymatic activity of MMPs is an important regulator of CNC cell migration. Endogenous MMP activity in vivo must be regulated in some way by naturally occurring inhibitors to limit the degree of proteolysis. The next chapter will evaluate the expression and potential function of natural inhibitors of MMPs, TIMPs, during early chick embryonic development and CNC cell migration.

58 Table 3-1

Effect of KB8301 Injection on MMP Activity"

46 pmoles Treated Group 92 pmoles r reated Group Experiment DMSO/Sham KB8301/Sham DMSO/Sham KB 8 301/Sham 1 0.941 0.820 1.060 0.778 2 0.980 1.085 0.985 0.758 3 1.039 0.993 0.980 0.365 4 ND ND 1.048 0.857 Mean ± SD 0.987 ± 0.049 0.966 ±0.135 1.018 ±0.042 *0.690 ± 0.221 +In each experiment, 10 embryos were injected with DMSO or KJB8301 and then reineubated for 6 hr. Another 10 embryos were sham operated. Tissue segments were then collected and pooled and MMP activity measured as described in Materials and Methods. Data in Table 3-1 represents the ratio of MMP activity in DMSO-treated to sham embryos and the ratio of KB8301-treated to sham embryos obtained in each experiment. Mean ± SD was calculated by pooling the data obtained in the four experiments and a t test was performed. Raw data is provided in Appendix 1. ND, not determined. * p < 0.05.

59 Table 3-2

Effect of KB8301 Injection on Early Embryonic Development*

End Stage of Collected Embryos Total ■# of P E xperim ent T reatm ent fD ose Injection E m bryos 1 2 -1 2 + 13-13'* 1 4 - 1 4 + value (pm oles) Stage

DMSO 10- 22 5 15 2 t (23% ) (68% ) (9% ) 1 >0.05 K B8301 46 10- 28 10 17 i (36% ) (61% ) (4% ) DMSO 10- 19 3 14 2 t (16% ) (74% ) (11% ) >0.05 2 K B8301 92 10- 21 9 9 3 (43% ) (43% ) (14% ) DMSO 10 35 1 20 14 t (3% ) (57% ) (40% ) >0.05 3 K B8301 46 10 32 4 20 8 (13% ) (62% ) (25% ) DMSO 10 29 5 19 5 t (17% ) (66% ) (17% ) >0.05 4 K B8301 92 10 39 14 16 9 (36% ) (41% ) (23% ) DMSO 10+ 33 0 6 27 t (0% ) (18% ) (82% ) >0.05 5 K B8301 46 10+ 42 0 10 32 (0% ) (24% ) (76% ) DMSO 10+ 35 2 10 23 t (6% ) (29% ) (66% ) >0.05 6 K B8301 92 10+ 30 3 8 19 (10% ) (27% ) (63% ) ‘Embryos were unilaterally injected with DMSO or KB8301 at the 2nd somite level at Hamburger and Hamilton stage 10', 10, 10+, reincubated for 13 hr, and then were collected and staged again. Embryos were classified into three groups based on their developmental stage at the time they were collected. The number of KB8301-treated embryos at each end stage was compared with DMSO controls using a chi-square test to determine whether embryonic development was significantly different in KB8301-treated and control embryos. injected with an equivalent amount of carrier (DMSO) used for KB8301 delivery.

60 Table 3-3

Effect of KB8301 on CNC cell Migration When Normalized for Embryo Size

Ratio of CNC migratory distance between

Injection injected and noninjected side (Mean ± SD)+ P value

stage DMSO KB8301

10- 0.981 ±0.072 0.919 ±0.082 <0.05

10 0.987 ±0.081 0.903 ±0.107 <0.001

10+ 1.003 ±0.050 0.921 ±0.084 <0.001

differences in embryo size were normalized by calculating the ratio between the measured CNC migratory distance on the injected and the noninjected sides for DMSO-treated embryos and KB8301-treated embryos and then comparing the ratios using a t test. See Appendix 2 for raw data.

61 Table 3-4

Relationship Between CNC Migratory Distance and Developmental Stage at the Time of

Collection

Ratio of Migratory Distance Between Injected Injection End Stage and Noninjected Sides Stage (Mean ± SD)f DMSO KB8301 10 12" -12+ 0.93 ±0.10 *0.89 ±0.14 10 13" 13+ 1.00 ±0.08 *0.90 ±0.07 10 14" -14+ 0.99 ±0.04 *0.92 ±0.12 f Stage 10 embryos receiving an injection of DMSO or 92 pmoles of KB8301 were reincubated 13 hr, and CNC migratory distance was measured as described in Materials and Methods. The ratio of migratory distance between the injected and noninjected side for DMSO-treated or KB8301-treated embryos was calculated. A two-way ANOVA was performed using the DMSO or KB8301 treatment as the column variable and the embryonic end stage of development as the independent variable. Results showed that regardless of end stage, KB8301 decreased CNC migratory distance. * p < 0.001. See Appendix 3 for raw data.

62 Figure 3-1. Dorsal view of whole mount HNK-1 immunostained embryos sham operated, treated with DMSO, or treated KB8301. (A) Sham operated embryo. Cranial

NC (arrows) and CNC cells (arrowheads) emigrated out of neural tube (n) and migrated normally. Otic pits (o) were also HNK-1 positive. (B) DMSO-treated embryo. DMSO was injected in the right side at the 2nd somite level. Cranial NC (arrows) and CNC cells

(arrowheads) emigrated and migrated away from neural tube (n). No recognizable difference from sham operated embryos was observed. (C) KB8301-treated (92 pmoles) embryo. Cranial NC (arrows) and CNC cells (arrowheads) migrated out of the neural tube

(n). The distance CNC cells migrated away from neural tube (n) on the right side

(injected with KB8301) appeared less than in the left side (noninjected side). (D) KB8301 injected (184 pmoles) embryo. KB8301 was injected at the right side of the 2nd somite level. The embryo was curved by the KB8301 treatment and appeared younger. Some cranial NC cells (arrows) were observed but no immunostaining for CNC cells

(arrowheads) was observed. However, trunk NC cells (asterisks) migration appeared normal. Abbreviations: n, neural tube; o, otic pit.

Figure 3-2. DAPI nuclear staining pattern at the 2nd somite level in DMSO-treated and

KB8301-treated embryos. (A) DMSO-treated embryo. Little or no nuclear fragmentation or shrinkage was observed. (B) KB8301-treated (92 pmoles) embryo. No increase of nuclear fragmentation or shrinkage was observed compared with A. (C) KB8301-treated

(184 pmoles) embryos. Nuclear fragmentation or shrinkage was visibly evident compared with A and B. Arrowheads: ectoderm. Scale bar = 906 /tm in A, B, and C.

63 Figure 3-3. Measuring CNC migratory distance in DMSO-treated and KB8301-treated

(92 pmoles) embryos. Image of HNK-1 immunostaining and DAPI nuclear staining of the identical section were combined using IPLab software. Migratory distance starting from the dorsal apex (arrowheads) of neural tube to the leading edge of migrating NC cells along the basement membrane of ectoderm was measured as indicated by the yellow line. (A) A section at 2nd somite level from a DMSO-treated embryo. No significant difference of migratory distance was observed between the injected side (arrow) and the noninjected side. (B) A section at 2nd somite level from a KB8301-treated embryo. CNC migratory distance was shorter in the injected side (arrow) than in the noninjected side.

The myocardium is also HNK-1 positive. Abbreviations: fg, foregut; m, myocardium; n, neural tube. Scale bar = 100 jam in A and B.

64 Figure 3-1

Figure 3-2

A V \ \ - ' *' '*» \ ‘ n V*. n K ?; ' i '; \ ' L i m fg fg * /V m f - r - V- * * '5^ \. * - Figure 3-3

La Figure 3-4. Effect of 46 pmoles of KB8301 on CNC cell migratory distance when injected unilaterally into embryos at stages 10", 10, or 10+. The CNC migratory distance at the level of the 2nd somite was measured in both the injected and noninjected side as described in Materials and Methods. No significant difference in CNC migratory distance between injected and noninjected sides was observed in either DMSO or KB8301 injected embryos nor when comparing the average migratory distance of CNC cells in the

KB8301 injected side with the average distance in the DMSO injected side. See

Appendix 4 for raw data.

66 r r

J> Migratory Distance (pm) ^ > <^ & J> Figure3-4 # 67 age 10+ e g ta S Figure 3-5. Effect of 92 pmoles of KB8301 on CNC cell migratory distance when injected unilaterally into embryos at stages 10', 10, or 10+. The CNC migratory distance at the level of the 2nd somite was measured on both the injected and noninjected side as described in Materials and Methods. A significant difference in CNC migratory distance between injected and noninjected sides was observed in KB8301 injected embryos (p <

0.01) but not in DMSO injected embryos. When comparing the average migratory distance of CNC in the KB8301 injected side with that in the DMSO injected side, a significant decrease was also observed (p < 0.05). See Appendix 5 for raw data.

68 Migratory Distance (pm) Figure 3-5 Figure 69 Figure 3-6. Cross-sectional area of the neural tube in embryos treated unilaterally with

DMSO or 92 pmol.es of KB8301 measured at the level of the 2nd somite. No significant difference was observed between DMSO-treated and KB8301-treated embryos. See

Appendix 6 for raw data.

70 Neural Tube Area (mitt) 20000 10000 12500- 15000- 17500- 2500- 7500- 5000- - 0 - -] Figure3-6 71 CHAPTER FOUR

ROLE OF TIMP-2 AND TIMP-3 DURING EARLY

CARDIAC NEURAL CREST FORMATION AND

MIGRATION

72 INTRODUCTION

Neural crest (NC) cells are a cell population originating from the border between neural plate and ectoderm and this cell population segregates and migrates out from the dorsal midline of forming neural tube through an epithelial-to-mesenchymal transformation [56], After exiting into a cell-free space beneath the surface ectoderm, the

NC cells migrate peripherally to contribute to a variety of tissue and organ systems including craniofacial structures, the peripheral nervous system, tunica media of great vessels, heart, and others, etc. [60,89,123], The NC cells derived from the axial level between the mid-otic and 3rd somite migrate into pharyngeal arches 3, 4, and 6 where some of the NC cells develop into smooth muscle cells of aortic arch arteries, cranial nerve neurons, cartilage, and other connective tissue. A portion of these NC cells invade the outflow tract of heart to participate in the formation of aorticopulmonary septum separating the outflow tract into the aorta and the pulmonary artery [62,90], Therefore, the NC cells derived between the mid-otic and 3rd somite level are dubbed cardiac NC cells (CNC cells).

The control of NC cell migration has long been a subject of speculation and experimentation. To date, research has been focused on the developmental expression and interaction of extracellular matrix (ECM) components with NC cells. One of the essential steps for differential interaction between ECM and NC cells is remodeling of

ECM components to accommodate NC cell migration, which in other cells mainly

73 involves a family of zinc enzymes named matrix metalloproteinases (MMPs). MMPs play important roles in pathological processes including tumor cell metastasis and in normal developmental events like angiogenesis [93,124-126]. Members of this proteinase family possess several conserved features including a consensus sequence for binding zinc at the catalytic site and a propeptide responsible for the latency of the zymogen.

Although MMP expression is regulated by growth factors and cytokines, once they are synthesized the enzymatic activity of MMPs is mainly controlled in three ways: transforming zymogens into active enzymes, degrading of the enzymes [67], and inhibiting enzymatic activity by tissue inhibitors of MMPs (TIMPs).

The family of TIMPs consists of four members [127-131]. TIMPs-1, -2, and -4 are secreted as soluble proteins, whereas TIMP-3 is anchored to the ECM [132], Binding of the amino terminal end of each TIMP to the catalytic site of activated MMPs leads to a nonselective inhibition of the enzymatic activity [133,134], Studies show that TIMPs inhibit tumor metastasis in experimental animal models [23,111] and over-expression of

TIMP-1 and TIMP-2 reduces melanoma cell and smooth muscle cell invasion in vitro

[114,135], Paradoxically, studies also show that TIMP-2 participates in the activation of proMMP-2 (gelatinase A) by MMP-14 (membrane type MMP type 1, MT1-MMP).

Studies suggest that MT1-MMP binds the N-terminal of TIMP-2 while the C-terminal of

TIMP-2 recruits proMMP-2 from ECM. This trimeric complex interacts with neighboring

TIMP-2-free MT1-MMP which then cleavages the propeptide domain from proMMP-2, and thus initiates the activation of proMMP-2 [9], Other types of MT-MMPs have the similar ability in activating proMMP-2 [67], Although evidence exists that extracellular domain of MT1-MMP can lead to proMMP-2 activation in the absence of TIMP-2 [136],

74 TIMP-2 null mutant mice do not produce active MMP-2 in vivo suggesting TIMP-2 is required for this process [18].

Apart from inhibiting MMP activity and facilitating proMMP-2 activation, studies suggest TIMPs are involved in cell proliferation and apoptosis independent of MMP activity. For example, recombinant TIMP-2 with an alanine appended to its N-terminus loses inhibitory activity against MMPs but still stimulates fibroblast proliferation [137].

TIMP-2 induces a dose-dependent reduction in rat vascular smooth muscle cell proliferation while TIMP-3 overexpression in this cell line promotes apoptosis. Both of these results are not mimicked by a synthetic MMP inhibitor [135], TIMP-2 also stimulates mesenchymal growth during morphogenesis of the rat metanephros, which again is unlikely due to TIMP-2 inhibition of MMPs since synthetic MMP inhibitor has no mesenchymal growth action [138]. Furthermore, the signaling pathway of TIMP-2 has been associated with tyrosine kinase activity and cAMP-dependent protein kinase activity [139-141]. Based on these data, a TIMP-2 receptor independent of MMPs has been proposed.

The functional role of TIMPs is not well understood in embryonic development. The expression pattern of TIMPs have been studied during embryo implantation and fetal development (> day 10.5) of mice. During mouse embryo implantation, MMP-9 is expressed in trophoblast giant cells while several other MMPs and TIMPs are expressed in the undifferentiated stroma in the outer decidua [101], Injection of a MMP synthetic inhibitor or overexpression of TIMP-1 reduces decidual length and overall size of the embryo, and the embryo is displaced mesometrially. This suggests MMP activity and

TIMP function are important for embryo implantation [101]. In mouse embryos (> day

75 10.5), TIMP-2 is abundant in mesenchymal tissues surrounding developing epithelia such as connective tissues surrounding nasal and oral epithelia, in the diaphragm, pericardium, auricle, lung pleural, and maturing skeleton [142], while TIMP-3 is expressed in cartilage, skeletal muscle, myocardium, liver, and some glandular epithelia [143]. In embryonic day 13-15 mouse mandibles, TIMP-1 and TIMP-2 are primarily co-expressed with MMP-9 and collagenase-3 in the ossifying areas of the mandible while TIMP-2,

TIMP-3, and stromelysin-3 are expressed in the skeletal muscle of the tongue [3]. These findings indicate that TIMPs play important roles during implantation and in fetal development. However, we have little knowledge regarding the temporal and spatial expression pattern of TIMPs during the embryonic period, when major morphogenetic events occur during development.

In Chapters 2 and 3 ,1 have shown that the distribution pattern of MMP-2 correlates with early CNC cell migration and that a synthetic inhibitor of MMPs, KB8301, can inhibit CNC cell migration in vivo. In this study, the expression patterns of TIMP-2 and

TIMP-3 were examined to determine if TIMP expression correlated with CNC cell migration and whether exogenous TIMP-2 would enhance or inhibit CNC migration in vivo. Our data showed that TIMP-2 was uniquely expressed in early migrating CNC cells and then expanded to other mesenchymal cells in older embryos. In contrast, TIMP-3 expression was only found in notochord and was not related to early CNC cell emigration or migration. Injection of recombinant TIMP-2 significantly decreased CNC cell migration suggesting TIMP-2 restricts or limits the degree of CNC cell migration in vivo.

76 MATERIALS AND METHODS

RT-PCR of Chicken TIMP-3

White Leghorn fertilized chicken eggs (Hy-Vac, Adel, IA) were incubated at 38°C in a 60% relative humidity environment until reaching Hamburger and Hamilton stage 13

[73]. Embryos were collected at RNase-free condition and total RNA was isolated using

TriReagent (Sigma Chemical Co., St. Louis, MO) according to manufacturer’s direction.

A GeneAMP RNA PCR kit (Perkin Elmer, Branchburg, NJ) was used to reverse- transcribe RNA and amplify cDNA of chicken TIMP-3. Briefly, the reverse transcription reaction mixture (full volume of 20 pi) consisted of 1 pg of total RNA, 4 pi of 25 mM

MgCl2, 2 pi of 10X PCR buffer II, 1 pi of RNase inhibitor, 1 ul of MuLV reverse transcriptase, 1 pi of oligo d(T)16, and 2 pi each of dGTP, dATP, dTTP, dCTP. After 10 min of incubation at room temperature, RNA was reverse-transcribed at 42°C for 15 min in a GeneAMP PCR system 2400 machine (Perkin Elmer, Branchburg, NJ). The transcription was stopped by heating to 99°C for 5 min. The resulted cDNA of TIMP-3 was amplified in the same machine. The amplification mixture (full volume of 100 pi) contained 20 pi of reverse transcription reaction, 4 pi of 25 mM MgCL, 8 pi of PCR buffer II, 0.5 pi of AmpliTaq DNA polymerase, 30 pmoles each of chicken TIMP-3 forward and reverse primers, and the balance water. The forward TIMP-3 primer was

AGG CTC GAG TTA GGC GAA CA and the reverse primer was GTC CAA AGG AAG

ATC AGC CAC based on the published sequence (Genbank accession number M94531).

The expected PCR product length was 740 bp. After initialization at 95°C for 105 sec, the sample was amplified for 35 cycles (1 cycle consisting of 15 sec at 95°C and 30 sec at

77 60°C) followed by a final extension of 7 min at 72°C. The resultant TIMP-3 cDNA was subsequently cloned into pPCR-Script Cam SK(+) plasmid using a PCR-Scnpt Cam

Cloning kit (Stratagene, La Jolla, CA). Briefly, the PCR product was precipitated with

4.0 M ammonium acetate and 100% ethanol followed by dissolving the pellet in TE buffer (1.0 M Tris-HCl, 0.5 M EDTA, pH 8.0). Five hundred ng of purified PCR product was polished in a 13.3 pi reaction containing 1 pi of 10 mM dNTP mixture, 1.3 pi of 10X polishing buffer, and 1 pi of cloned Pfu DNA polymerase at 72°C for 30 min. Two hundred ng of the polished product was incubated with 10 ng pPCR-Script Cam SK(+) plasmid for ligation in a reaction containing one pi of pPCR-Script 10X buffer, 1 pi Srf I restriction enzyme, 1 pi of T4 DNA ligase, and 0.5 pi of 10 mM rATP at 12°C for 1 hr followed by heating to 65°C for 10 min. The plasmids were subsequently transformed into Epicurian Coli XL-1-Blue MRF’ Kan supercompetent cells. TIMP-3 cDNA was isolated using a modified mini alkaline-lysis/PEG precipitation procedure [144] and was confirmed by DNA sequencing.

In Situ Hybridization

Digoxigenin-labeled antisense and sense riboprobes for chicken TIMP-3 and

TIMP-2 (TIMP-2 cDNA was a generous gift from Dr. James P. Quigley, Scripps

Research Institute, La Jolla, LA) [145] were prepared using in vitro transcription method

(Genius 4 RNA Labeling Kit; Roche Molecular Biochemicals, Indianapolis, IN) after linearizing the plasmids. The length of TIMP-2 and TIMP-3 riboprobes were 660 and 760 bp respectively. Stage 8-14 chicken embryos were fixed in 4% paraformaldehyde overnight, rinsed in phosphate-buffered saline (PBS), and subjected to in situ

78 hybridization as described in Chapter 2. The hybridization and washing temperatures for

TIMP-2 and TIMP-3 were 70°C and 60°C, respectively. Hybridization by sense TIMP-2

and sense TIMP-3 riboprobes was not detected in embryos at any stage under these

conditions. Some TIMP-2-hybridized embryos were immunostained as whole mounts

with HNK-1 as described in Chapter 3. Embryos were embedded in TBS tissue freezing

medium (Triangle Biomedical Sciences, Durham, NC) and serially sectioned on a

cryostat for further examination.

MMP Enzymatic Activity Assay

Stage 10 chick embryos were bilaterally injected with 4.6 ng of human

recombinant TIMP-2 (Chemicon International, Temecula, CA) between the otic pit and

2nd somite level. Injected embryos were then re-incubated for 6 hr. Control embryos

were injected with same volume of Ringer’s solution or sham operated. Tissue segments

were then collected and assayed for MMP activity as described for KB8301 in Chapter 3.

TIMP-2 Injection and CNC Cell Migratory distance Assay

A total of 4.6 ng of human recombinant TIMP-2 was injected unilaterally at the

2nd somite level of stage 10 chick embryos. Control embryos were injected with the

same volume of Ringer’s solution. The embryos were then incubated for an additional 13

hr until they reached stage 13-14 and then collected, fixed in 4% paraformaldehyde, and

rinsed with PBS. Embryos were then immunostained with HNK-1, embedded in JB-4

plastic, and serially sectioned. The degree of CNC cell migration was assessed as

described in Chapter 3.

79 RESULTS

Expression of TIMP-2 and TIMP-3 in Chick Embryos

To determine whether the expression of TIMP-2 and TIMP-3 in chicken embryos correlated with MMP-2 expression and early CNC cell migration, in situ hybridization studies for TIMP-2 and TIMP-3 mRNA were performed on stages 8 to 14 chicken embryos. Prior to stage 10, TIMP-2 mRNA was detected within the midbrain and hindbrain neural tube (Fig. 4-1A). Beginning stage 11, the expression of TIMP-2 mRNA within the neural tube gradually decreased except in 5 (R5) and 6 (R6), where TIMP-2 mRNA levels increased until stage 14 (Fig. 4-lB,C,D). Migrating CNC cells also began to express TIMP-2 message at stage 12 and continued to express TIMP-2 mRNA as they entered the pharyngeal arches (Fig. 4-lB,D-G,I,J). By this time (stage 14),

TIMP-2 mRNA expression expanded to include most of the mesenchyme found in pharyngeal arches and the endocardium of heart. Colocalization with HNK-1 immur.ostaining showed that TIMP-2 was only expressed within the subset of CNC cells adjacent to the ectoderm but not in all CNC cells (Fig.4-ID,E, F,G). In addition, TIMP-2 expression levels appeared to be higher in pioneering (leading CNC cells) than in trailing

CNC cells (Fig. 4-lF,G). No hybridization was detected with a sense probe for TIMP-2

(Fig. 4-1H).

TIMP-3 was first detected in the notochord at stage 8 and was continually expressed there through stage 14 (Fig.4-1K,L,M). Beginning stages 10+ to 11, TIMP-3

80 mRNA was also detected in myocardium of the outflow tract (Fig 4-1K). By stage 13,

TIMP-3 mRNA expression expanded to include the endocardium as well as the endothelium of developing big vessels. By stage 14, TIMP-3 was also expressed in the pharyngeal arch mesenchyme but the intensity of antisense probe hybridization was less than in the notochord or heart (Fig. 4-1M). TIMP-3 was not detected in neural tube, CNC cells, or paraxial mesoderm of embryos before stage 14 (Fig. 4-lK,J,M). No hybridization was detected with sense probe for TIMP-3 (Fig. 4-IN).

MMP Enzymatic Activity After Injection of TIMP-2

To determine if microinjection of human recombinant TIMP-2 inhibited MMP enzymatic activity in vivo, MMP activity in embryo was measured 6 hr after TIMP-2 injection. Bilateral injection of 4.6 ng of TIMP-2 significantly reduced the level of MMP activity in treated embryos compared to Ringer’s -treated embryos (Table 4-1).

Effect of TIMP-2 Injection on Chicken Embryonic Development

Chicken embryos were unilaterally injected at 2nd somite level with Ringer’s solution or TIMP-2 at stage 10 and collected after 13 hr of re-incubation. Among the 28 embryos injected with TIMP-2, two died, while all 22 embryos injected with Ringer’s solution survived. In both groups, surviving embryos had no discernible morphological defects or change in size based on visual observation. The rate of embryonic development based on the embryonic stage at the time of collection between TIMP-2-treated and

Ringer’s-treated groups was not significantly different (Table 4-2, chi-square test p >

0.05). In addition, there was no observable difference in DAPI neclear staining between

81 Ringer’s-treated or TIMP-2-treated embryos, suggesting that effects of TIMP-2 microinjection on CNC cell migration was not due to differences in cell death (Fig. 4-

2A,B).

Effect of TIMP-2 Injection on CNC Cell Migration

The migratory distance of CNC cells in injected and noninjected sides of TIMP-2- treated and Ringer’s-treated embryos was measured from the mid-apex of the neural tube to the leading edge of the migrating CNC cell population (Fig. 4-3A,B). There was a small but significant decrease in CNC cell migratory distance in the injected side compared with that in the noninjected side of TIMP-2-treated embryos. There also was a significant difference in CNC cell migratory distance between the injected side of

Ringer’s-treated embryos and the injected side of TIMP-2-treated embryos (both p <

0.05). No difference between the Ringer’s-injected and noninjected sides was observed

(Fig. 4-4).

To exclude the possibility that measured differences in migratory distance could be attributable to TIMP-2 effects on embryo size, the ratio of CNC migratory distance between the injected side and noninjected side in TIMP-2-treated embryos and Ringer’s- treated embryos was compared. Results still showed that there was a significant decrease in migration in TIMP-2-treated embryos compared to controls when embryo size was taken into account (Table 4-3).

82 DISCUSSION

Studies show that MMP activity and its regulation by TIMPs are important mediators of epithelial-mesenchymal transitions and cell migration during embryonic development [41,146,147]. NC cells are one of the most extensively migrating and invasive population of cells found in embryos. In this study, I showed for the first time that TIMPs are important mediators of NC morphogenesis as evidenced by (1) the unique and limited expression of TIMP-2 mRNA in CNC cells during their early migration and

(2) the inhibition of CNC migration in embryos injected with TIMP-2.

TIMP-2 Expression

The earliest stage TIMP-2 mRNA transcripts were detected was at stage 9-10 in the lumenal side of neural tube but expression became limited to rhombomeres 5 and 6

(R5/6) by stage 11. The significance of TIMP-2 expression in R5/6 is unknown. R5 is regarded by some investigators as a region of the neural tube that gives rise to NC cells which are subsequently eliminated through apoptosis [152,153], However, other studies show that at least some of the NC cells derived from this axial level survive, migrate rostral and caudal to the region, and enter pharyngeal arches 2, 3, and 4 [154-

156], TIMP-2 can regulate cell proliferation and apoptosis indirectly by mediating MMP- dependent events (e.g., growth factor activation and turnover, ligand release and receptor turnover, etc.) or possibly through MMP-independent TIMP-2 receptors [135,157]

[114,139,140,158], Therefore the restricted expression of TIMP-2 in R5/6 may somehow

83 be involved in regulating the complicated morphogenesis of NC cells generated at this axial level.

TIMP-2 Expression in CNC

TIMP-2 mRNA began to be expressed in CNC cells at stage 12 when the pioneering CNC cells had emigrated and migrated some distance from the neural tube.

No expression of TIMP-2 was detected in mesencephalic and trunk NC cells. Why

TIMP-2 is expressed in NC ceils derived from this axial level but not elsewhere along the axis is unknown. TIMP-2 expression in CNC cells may be intrinsic to this population of

NC cells or induced/repressed by local signals. It is well established that gene expression is controlled by positional and segmental genes such as the paralog group 3, 4, and 5 genes that dictate regional specificity to neural tube and NC cells generated along the anterior/posterior body axis [159], Transplantation studies show that neither mesencephalic nor trunk NC cells are capable of generating mesenchyme competent to replace CNC with regard to cardiac septation. This suggests that CNC cells are unique

[160], Therefore, TIMP-2 expression in CNC cells may reflect a positionally-restricted

(axial-dependent) lineage specification of NC cells.

The restricted expression of TIMP-2 in CNC cells may also occur as a result of regionally-restricted regulatory signals emanating from adjacent tissues like the ectoderm and the neural tube. For instance, TIMP-2 expression may be induced in CNC cells by ectoderm (such as members of the TGF-6 family which are capable of inducing TIMP-2 in many cell types) [161-165] [166]. In this study, CNC cells immediately adjacent the ectoderm expressed TIMP-2 mRNA but those not adjacent the ectoderm did not express

84 TIMP-2 mRNA. Alternatively, TIMP-2 expression in emigrating and newly formed CNC could be suppressed by neural tube signals. As CNC cells migrate away, this repressive influence is lost and TIMP-2 expression increases. However, this is not likely given the observation that after stage 12 newly-formed and migrating CNC cells adjacent neural tube also expressed TIMP-2 and that TIMP-2 mRNA was not detected in trunk or cranial

NC cells after they migrated from the neural tube. Neural crest, neural tube, or ectodermal transplantation experiments would be needed to determine if any of these possible scenarios could account for the limited expression of TIMP-2.

TIMP-3 Expression

Of the two TIMPs examined, TIMP-3 was the earliest to be expressed. TIMP-3 was specifically expressed in the notochord beginning stage 8. Expression of TIMP-3 gradually widened to include the myocardium and endocardium, and then expanded to include the endothelium of big vessels and the pharyngeal arch mesenchymes in older embryos. Like other TIMPs, TIMP-3 is capable of blocking the enzymatic activity of most of MMPs tested to date [67]. The unique expression of TIMP-3 in the notochord suggests that TIMP-3 may participate in the development and remodeling of the floor plate, adjacent endoderm, and sclerotome by modulating MMP including MMP-2, which is also found here (Described in Chapter 2). NC cell migration is excluded from this area due in part to the presence of extracellular chondroitin sulfate proteoglycan [148,149].

However, expression of TIMP-3 in the notochord could also limit NC invasion of this area by blocking MMP activity needed for invasion or degradation of inhibitory molecules.

85 After stage 13, TIMP-3 mRNA expression expanded to include the mesenchyme of pharyngeal arches but it was more prevalent in the endothelium of aortic arch arteries.

Aortic arch arteries develop from cords of endothelial precursors that are surrounded by invading NC-derived mesenchyme and these early vessels undergo major remodeling during embryonic development [60,150], TIMP-3 is an important mediator of angiogenesis and vascular smooth muscle proliferation and apoptosis [135,151],

Accordingly, TIMP-3 could be involved in modulation events needed to establish the adult pattern of great vessels from the aortic arch primordia. Regardless, the temporal and spatial distribution of TIMP-3 expression makes it unlikely to have a role in CNC emigration and then early migration.

Functional role of TIMP-2

Based on the in situ hybridization data, TIMP-2 is unlikely to be involved in epithelial-mesenchymal transition and emigration of CNC cells that occur at stages 10-

11. Rather, the functional role of TIMP-2 may be to modulate subsequent migration of

CNC cells. Exogenous TIMP-2 significantly reduces the invasive capacity of melanoma cells or glioblastoma cells within a three-dimensional type I collagen lattice [167,168],

Transfection of TIMP-2 into gastric carcinoma cells significantly reduces their invasiveness in vitro and in vivo [169], In this study, injection of a small amount of

TIMP-2 into the CNC migratory pathway reduced MMP activity and decreased the distance that CNC cells migrated. The decrease in migratory distance was not attributable to changes in development or growth rate of the embryos. This shows that TIMP-2 is an important mediator of NC migration, possibly by modulating MMP activity.

86 In order to migrate, CNC cells must penetrate ECM barriers and then move from one point to another. Morphological studies show that basement membrane-associated

ECM is lost in areas occupied by invading cephalic NC cells [80]. As described in

Chapter 2, MMP-2 is deposited in this basement membrane. MMP-2 can degrade type IV collagen, fibronectin, and other known components of this basement membrane. Studies also show that proMMP-2 can not be activated in vivo in the absence of TIMP-2 [18].

Interestingly, pioneering CNC cells had more mRNA than trailing CNC cells suggesting there were more TIMP-2 molecules and hence more active MMP-2 associated with the leading edge of invading CNC cells. Active remodeling of the basement membrane- associated ECM by pioneering CNC cells could influence the migratory behavior of trailing CNC cells as the migratory pattern and developmental fate of late migrating NC cells have been shown to depend on early migratory NC cells [81]. Therefore, the TIMP-

2 produced by CNC cells may play a major role in mediating CNC invasion of ECM by facilitating the activation of proMMP-2 sequestered in ectodermal basement membrane.

Synthesis of particular TIMP-2 appears to be important for MMP-2 activation and cell migration. Studies show that low concentrations of TIMP-2 (i.e., the molar ratio of

MT1-MMP to TIMP-2 ranging from 3:1, 2:1, and 3:2) promotes the activation process of proMMP-2. High concentrations of TIMP-2 (i.e., the molar ratio of MT1-MMP to TIMP-

2 being 1:1 or with an excess of TIMP-2) inhibit the activation process, as increasing

TIMP-2 levels begin to saturate MT1-MMP thereby blocking proMMP-2 activation and

MMP-2 activity directly [170]. In this study, TIMP-2 was uniquely expressed in migrating CNC cells. Therefore, if TIMP-2 modulates MMP-2-dependent migration,

TIMP-2 levels must be appropriate for promoting MMP-2 activation necessary for CNC

87 invasion and migration. However, if TIMP-2 concentrations are increased locally by microinjected TIMP-2, one would predict a decrease in MMP activity and CNC migration as observed in this study. Therefore, future studies and their interpretation must take into consideration the biphasic effect TIMP-2 can have on active MMP levels in vivo.

Injection of TIMP-2 may also decrease CNC migration by perturbing CNC motility. Cell migration and invasion of the ECM involves formation of focal contacts in lamellopodia and invadopodia. Focal contacts are specialized cell membrane/substrate contact sites containing cell membrane integrins linking ECM components with the cytoskeleton. Lamellopodia and invadopodia are distinct cell surface protrusions found at the leading edges of invasive cells that interact with the ECM via focal contacts and are considered the main organelle responsible for cellular locomotion and invasion. MMP-2,

MT1-MMP, and gelatinase activity colocalized within focal contacts of both lamellopodia and invadopodia [21,86,171,172], In addition, studies suggest that the

TIMP-2 in lamellopodia and invadopodia mediates gelatinolytic activity within focal contacts [86], Exogenous TIMP-2 or its overexpression inhibits the degradation of gelatin substrates by migrating cells, increases cell spreading, and inhibits cell motility of several cell types [173] [11,114], These observations suggest that TIMP-2 regulation of MMP activity in focal contacts of CNC likely plays an important role in mediating cell/ECM adhesion and migration. By injecting TIMP-2 into NC migratory pathways, TIMP-2 may have increased CNC cell adhesion to ECM and reduced CNC cell motility.

88 TIMP-2 and Embryonic morphogenesis

TIMP-2 has been shown to be involved in organ morphogenesis. Incubating 2

ug/ml of human recombinant TIMP-2 with E l3 rat kidney inhibits branching of ureteric

buds and alters the deposition of its basement membrane. These effects are thought to be

due to TIMP-2 inhibition of MMP activity as these effects are reproduced by synthetic

MMP inhibitors [138], Incubating E9 rat embryos with TIMP-2 or synthetic MMP

inhibitors decreased embryo growth but produced no obvious malformations within the

developmental period examined [174], While these studies show excess TIMP-2 can

cause developmental changes, the TIMP-2-treated embryos in this study did not show

any noticable morphological defects or changes in developmental rate or size alteration.

The lack of a teratogenic effect could be due to the dose of TIMP-2 applied in

experiments. The concentration of injected TIMP-2 in this study was 0.5 /tg/rnl that was

far less compared with that used in other studies (usually more than 1 jUg/ml in culture

media) and a single 4.6 ng injection of TIMP-2 was used. Moreover, the TIMP-2 levels

likely decreased over time due to diffusion, turnover, and increasing of embryo size.

While TIMP-2 injection had no discemable effect other than decreasing CNC migratory

distance, developmental consequences could still appear with prolonged incubation or

with higher concentrations of TIMP-2.

TIMP-2 null mice have been generated and these mutant mice show no obvious

developmental abnormalies [18]. As there are more than 20 types of MMPs, many with

overlapping substrate specificities, and at least four types of TIMPs, the loss of TIMP-2

and MMP-2 could be compensated for by these other MMPs and inhibitors. Therefore the

specific function of TIMP-2 and MMP-2 in CNC morphogenesis and migration will be

89 difficult to assess in vivo using null mouse model. By using an in vivo chick model, this study provides the first direct evidence that TIMP-2 mediates CNC cell migration.

In conclusion, while TIMP-3 may be involved in later stages of NC migration and aortic arch remodeling events, based on the in situ hybridization studies it is not likely to play a role in CNC emigration or early migration. In contrast, TIMP-2 is specifically expressed within CNC cells adjacent the ectoderm and appears to be continually expressed within pharyngeal arches of older embryos. Injection of TIMP-2 into the CNC pathway prior to their emigration reduced MMP activity in embryos and decreased the distance CNC cell migrated from neural tube on the injected side. This showed TIMP-2 levels are an important factor in mediating NC migration. Why TIMP-2 expression is restricted to CNC cells and not in other migrating NC cells is unclear and its precise function is unknown. Answers to these questions will require further study.

90 Table 4-1

Effect of TIMP-2 Injection on MMP Enzymatic Activity!

Experiment Ringer’s/Sham TIMP-2/Sham 1 0.957 0.794 2 1.023 0.829 3 0.967 0.607 Mean ± SD 0.982 ±0.036 *0.743 ±0.119 t In each experiment, ten HH stage 10 embryos were injected with Ringer’s solution or TIMP-2 and re-incubated for 6 hr. Another 10 embryos were sham operated. Tissue segments were then collected and pooled into their respective group and MMP activity measured as described in Chapter 3. Data in Table 4-1 represents the ratio of MMP activity in Ringer’s solution-treated or TIMP-2-treated to sham embryos obtained in each experiment. Mean + SD was calculated by pooling the data obtained in the 3 experiments and a t test was performed. * p < 0.05. See Appendix 7 for raw data.

91 Table 4-2 Effect of TIMP-2 Injection on Embryonic Development! — Stage of Collected Embryos Treatment Embryos 12-12+ 13'-13+ 14 _14+ P Value Number 1 18 3 Ringer’s saline 22 (5%) (82%) (14%) >0.05

Ai 24 1 TIMP-2 26 (4%) (92%) (4%) tEmbryos unilaterally injected at the 2nd somite level with Ringer’s saline or TIMP-2 at stage 10 were collected after 13 hr of additional incubation, staged again, and fixed. These embryos were then divided into three groups based on their end stage of development when collected. Data in Table 4-2 represents the number and percentage of embryos in each group. The number of TIMP-2-treated embryos in each group was compared to that of Ringer’s solution-treated controls using a chi-square test to determine if the rate of embryonic development was altered by TIMP-2 injection.

92 Table 4-3

Effect of TIMP-2 Injection on CNC Migration After Normalizing for Potential

Differences in Embryo Sizef

Treatment CNC Migration (Injected Side/Noninjected Side) Ringer’s saline 1.003 ±0.068 TIMP-2 *0.897 ±0.075 t Normalizing for differences in embryo size was accomplished by calculating the ratio between the measured CNC migratory distance on the injected and the noninjected sides for Ringer’s-treated and TIMP-2-treated embryos and then comparing the ratios using a t test. * p < 0.05. See Appendix 8 for raw data.

93 Figure 4-1. TIMP-2 and TIMP-3 rnRNA expression during early CNC morphogenesis,

(A) Whole mount TIMP-2 in situ hybridization of a stage 10- embryo, ventral view.

TIMP-2 rnRNA was expressed in neural tube (arrows). (B) Whole-mount TIMP-2 in situ hybridization of a stage 13 embryo, dorsal view. Cells in 5/6 (arrow) and

CNC cells (arrowheads) expressed TIMP-2 rnRNA. (C) A cross section of the embryo shown in B at rhombomere 5 level. Neural tube cells (arrows) and endocardial cells

(arrowheads) expressed TIMP-2 rnRNA. (D) A cross section of the embryo shown in B at rhombomere 6 level. Neural tube cells (arrows), migrating CNC cells (arrowheads), and endocardial cells expressed TIMP-2 rnRNA. (E) HNK-1 immunostaining of the same section shown in D. HNK-1 bound to the migrating CNC cells. Comparing E with D,

only a portion of CNC cells adjacent to ectoderm (arrowheads) expressed TIMP-2

rnRNA. The CNC cells adjacent the mesoderm (arrows) did not express TIMP-2

message. (F) A cross section of the embryo shown in B at rhombomere 7 level. Migrating

CNC cells expressed TIMP-2 rnRNA. The leading CNC cells (arrowheads) expressed

more TIMP-2 message than the trailing CNC cells (arrows). (G) HNK-1 immunostaining

of the same section shown in F. HNK-1 bound to CNC cells (arrowheads) and

myocardial cells (arrows). (H) A stage 13 embryo treated with TIMP-2 sense riboprobe

as a control. No hybridization was detected. (I) A cross section of the embryo shown in B

at the 2nd somite level. Migrating CNC cells (arrowheads) expressed TIMP-2 rnRNA. (J)

HNK-1 immunostaining of the same section shown in I. HNK-1 bound to migrating CNC

cells (arrowheads) and myocardial cells (arrows). (K) A cross section of a stage 11

embryo hybridized with TIMP-3 antisense riboprobe. TIMP-3 rnRNA was expressed in

notochord (arrow) and myocardium (arrowheads). (L) Whole mount TIMP-3 in situ

94 hybridization of stage 9+ embryo, ventral view. TIMP-3 mRMA was only detected in notochord (arrowhead). (M) Whole mount TIMP-3 in situ hybridization of a stage 14 embryo, side view. TIMP-3 mRNA was expressed in paraxial mesoderm (pm), pharyngeal arch mesenchyme (arrow), endocardium and myocardium of heart (h), and notochord (arrowhead). (N) A stage 12 embryo treated with TIMP-3 sense riboprobe. No hybidization was detected. Abbreviations: h, heart; n, neural tube; m, myocardium; o, otic; pa, pharyngeal arch; pm, paraxial mesoderm, so, somite. Scale bar = 100 /xm for C,

D, E, F, G, I, J, K.

95

Figure 4-2. DAPI nuclear staining of sections from Ringer’s-treated and TIMP-2-treated embryos. (A) DAPI nuclear staining of a cross section at the 2nd somite level from a

Ringer’s-treated embryo. Few nuclear fragmentation or shrinkage was observed. (B)

DAPI nuclear staining of a cross section at the 2nd somite level from a TIMP-2-treated embryo. No increase of nuclear fragmentation or shrinkage was observed compared with

A. Arrowheads: ectoderm. Scale bar = 609 /am in A and B.

Figure 4-3. Measurement of CNC migratory distance on cross sections from Ringer’s- treated or TIMP-2-treated embryos. CNC migratory distance was indicated as the yellow line. (A) Combined images of HNK-1 immunostaining and DAPI nuclear staining from the same cross section of a Ringer’s-treated embryo. Migratory distance was measured beginning at the neural tube apex (arrowhead) and ending at the leading CNC cells along the basement membrane of ectoderm in the injected side (arrow) and the noninjected side.

(B) Combined images of HNK-1 immunostaining and DAPI nuclear staining from the same cross section of a TIMP-2-treated embryo. Migratory distance was measured in the injected side (arrow) and the noninjected side as in A. Abbreviations: f, foregut; n, neural tube. Scale bar = 100 /mi in A and B.

97 Figure 4-2

Figure 4-3

p Figure 4-4. Effect of TIMP-2 injection on CNC cell migration when injected unilaterally into embryos at stage 10. After re-incubation for 13 hr, embryos were immunostained with HNK-1 and the migratory distance at the level of the 2nd somite was measured on both the injected and noninjected side as described in Chapter 3. A significant difference in CNC migratory distance between the injected and the noninjected sides was observed in TIMP-2-treated embryos (p < 0.05) but not in Ringer’s controls. A significant difference in CNC migratory distance was also observed between the injected sides of

Ringer’s and TIMP-2-treated embryos (p < 0 05). See Appendix 9 for raw data.

99 Migration Distance (pm) Figure 4-4 Figure •iejc-k 100 GENERAL DISCUSSION

Cell migration and tissue remodeling are two central themes during embryonic development. For cells to migrate, they must acquire motility, generate fractional forces and invade ECM in order to relocate themselves. Numerous studies show that the versatile use of different proteinase systems with a variety of localization mechanisms and cleavage targets is an integral feature of migrating cells in both physiological and pathological processes (Reviewed by Ellis and Murphy, 2001) [175], NC cells are one of the most extensive migrating cell populations in the embryo and their derivatives are indispensable for development of many organs. Therefore, studying the mechanisms oi'

NC migration is fundamental to the understanding of normal embryonic development as well as congenital defects. The plasminogen activator system has been shown to be involved in NC cell migration [38,39,102,176], but transgenic null mice lacking plasminogen activator or urokinase receptors are viable and fertile suggesting other proteinase systems, particularly MMPs, may be involved in NC cell migration [177,178],

In this study, 1 provide evidence that MMPs are involved in modulating NC cell migration. I found that the spatial and temporal expression of MMP-2 and TIMP-2 correlated positively with CNC cell migration and that injecting MMP inhibitors reduced

CNC cell migration in vivo. While MMP activity was correspondingly decreased in the injected embryos, the exact cause for the reduction of CNC migration was unclear.

MMP-2 protein was found in the basement membrane of the ectoderm and neural folds prior to CNC cell emigration. Injection of the MMP inhibitors may have decreased CNC cell emigration because MMP activity necessary for penetrating basement membrane was

101 blocked. Moreover, just prior to CNC cell emigration, the basement membrane is discontinuous beneath NC progenitors [77] and MMP-2 immunostaining was also discontinuous suggesting local depletion. These observations support the idea that active

MMPs, like MMP-2, may have an important role in CNC cell emigration that would be reflected as a decrease of the distance CNC cell migration in my assays.

Another possible mechanism by which MMP inhibitors could decrease the distance CNC cell migrated was that the inhibitors decreased CNC cell motility and invasiveness. Once out of the neural tube, CNC cells must exhibit motility and traverse and invade ECM. Studies show that ectodermal basement membrane-associated ECM is lost in the areas occupied by invading NC cells [80], In this study, I showed that while

NC cells did not synthesize MMP-2, migrating NC cells encountered MMP-2 protein within the ectodermal basement membrane that was synthesized by the paraxial mesoderm. CNC cells could employ two mechanisms for recruiting extracellular MMP-2 to the CNC cell surface. One mechanism involves generation of a MT-MMP/TIMP-2 complex as described in the General Introduction. Support for this scenario is provided by my observation that TIMP-2 was specifically expressed by CNC cells soon after they emigrated from the neural tube. While MT3-MMP expression in CNC cells could contribute to the formation of this complex, in situ hybridization studies showed MT3-

MMP was not expressed at this stage. Therefore, if such a complex is formed, MT1-

MMP is the most likely MT-MMP candidate as several cell types have been shown to form a ternary complex using this MT-MMP [9,179,180]. Testing this hypothesis will require that the chicken homolog of MT1-MMP be identified.

102 Another possible recruiting mechanism involves the integrin a vB3. a vB3 is expressed by NC cells and injection of a vB3 antibodies inhibits NC migration [181] [34]. a vB3 is capable of binding proMMP-2 [29,182], Studies suggest that binding of MMP-2 via this integrin brings proMMP-2 to cell surface where it can be activated. In fact, a vB3 integrin binding to proMMP-2 promotes the full activation of MMP-2 after MT1-MMP initiates the process [183], However, we should also consider the possibility that proMMP-2 could serve as an adhesion molecule with a potential role in mediating turnover of the adhesion complex once the proMMP-2 is activated. Collectively, my observations showing MMP-2 was associated with the cell surfaces of CMC cells and that

inhibiting MMP activity reduced CNC cell migration support the postulate that MMP-2 plays an important role in CNC cell motility and invasiveness.

In addition to potentially modulating CNC emigration and ECM invasiveness,

MMP activity may expose biological active “cryptic” sites contained within the ECM to

CNC cells and thereby regulating CNC migration. Studies show that cleavage of laminin-

5 by MMP-2 induces breast epithelial cell migration [14] while proteolytic fragments of

type IV collagen are essential for endothelial cell migration during angiogenesis in vivo

[184], Given that laminin and type IV collagen are abundant within CNC migratory

pathways, cryptic fragments of these molecules generated by MMP at the leading edge of

migratory CNC cells could have a very important role in mediating CNC migration.

Furthermore, MMP activity may influence CNC migration by regulating the cleavage of

cell surface receptors and ligands. CD44, a major cell-surface receptor for hyaluronate, is

expressed in the pre-migratory and migratory cranial NC cells of chick embryos [185],

MT1-MMP is capable of shedding CD44 and subsequently promotes cell migration

103 [186]. MMP activity can also release growth factors from ECM and regulate cell behavior [187], Therefore, inhibiting MMP activity in the embryo could disrupt these important regulatory factors during CNC emigration and migration.

Perhaps the most intriguing observation is the unique and highly restricted expression of TIMP-2 in CNC cells during their early migration and only in a subset of these cells. Because TIMP-2 appears to be expressed for proMMP-2 activation, the most probable role for TIMP-2 expression in CNC cells is to recruit, activate and modulate

MMP-2 activity. Pioneering CNC cells expressed more TIMP-2 mRNA than the trailing cells where the bulk of ECM degradation would occur as the CNC population traverses the ECM. Therefore, pioneering CNC cells would be the first to encounter MMP-2 and as such would be expected to be the site of higher MMP-2 activity and ECM degradation. If

TIMP-2 is required for activation of MMP-2, why would injecting TIMP-2 decrease

CNC migration? Studies show that low concentrations of TIMP-2 facilitate proMMP-2 activation but high concentrations block MMP-2 activity as more of the MT-MMPs as well as active MMP-2 are inhibited[ 170]. My observation showing that microinjection of exogenous TIMP-2 decreased both the distance CNC cells migrated as well as MMP

activity in vivo supports the postulate that CNC cells likely regulate the activation of

proMMP-2 by synthesizing and releasing appropriate amounts TIMP-2 needed for their

migration. However, this scenario would not apply to cranial and trunk NC cells since

TIMP-2 mRNA was not detected in these cells during their early migration. Therefore,

cranial and trunk NC cells may employ other MMPs or other proteolytic systems like

plasminogen activator/plasmin system or ADAMs for their migration.

104 SUMMARY

The temporal and spatial distribution of MMPs and TIMPs described in this thesis suggests these molecules have a role in NC morphogenesis. Directly inhibiting MMP activity in normal embryos using a synthetic MMP inhibitor or one expressed by CNC cells, provides direct evidence that MMP activity is an important mediator of CNC cell migration. It is conceivable that environmental influences or genetic alterations causing inappropriate expression of MMPs or their inhibitors could lead to NC-related congenital defects.

Studies of knockout mice lacking functional proteases show that embryonic development including development of NC-dependent organs, is remarkably plastic in circumventing the loss of proteolytic activity. While precise developmental events leading to the formation of specific structures and organs have not usually been examined in these null animals, studies show that evidence suggests developmental pathways leading to viable and fertile animals can be quite different from their wild-type littennates. The MMP system provides migratory CNC cells with an alternate proteolytic mechanism to the plasminogen activator/plasmin system. Now that knockout mice for plasminogen activator/plasmin system have been generated as well as for some of the

MMPs and TIMPs, specific crosses generating multiple knockouts between these two proteolytic systems may further elucidate the roles of proteinases in NC morphogenesis.

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131 Appendix

132 Appendix 1 Table 3-1. Effect of KB8301 Injection on MMP Activity QxU/jtig DNA/20 min) Test Dose of Sham DMSO -Treated KB 8301-Treated KB8301 (pmoles) 1 92 167.0 177.0 130.0 2 92 66.0 65.0 50.0 3 92 197.0 193.0 72.0 4 92 84.0 88.0 72.0 5 46 166.9 157.1 136.9 6 46 136.6 133.8 148.2 7 46 112.5 116.9 111.74

133 Appendix 2

Table 3-3. Effect of KB8301 on CNC Migration When Normalized for Embryo Sizet

St. 10- in ./noninj. St. 10 in j./noninj. Stl0+ in ./noninj. Embryo # DM KB DM KB DM KB 1 0.95 0.95 0.98 0.86 0.99 0.94 2 0.99 0.79 0.95 0.86 0.97 0.92 3 0.98 1.00 1.02 0.81 1.10 0.97 4 0.94 0.88 0.98 0.91 1.02 1.03 5 0.98 0.99 0.99 0.78 1.03 1.12 6 1.01 0.90 1.00 0.95 1.04 1.01 7 0.97 0.85 1.01 0.87 1.08 0.96 8 1.06 0.76 1.03 0.81 1.14 0.99 9 0.86 0.93 1.02 0.97 1.05 0.90 10 0.95 0.86 0.92 0.91 1.00 0 86 11 1 00 0.78 0.98 0.97 0.95 0.93 1.10 0.91 12 0.83 1.07 1.00 C.77 '| 13 1.07 0.94 0.99 0.94 0.94 0.83 14 1.10 0.96 0.96 0.79 1.00 0.75 15 1.02 0.91 1.06 0.93 1.04 0.94 16 1.03 0.86 1.20 0.98 0.95 0.98 17 0.97 0.83 0.90 0.62 0.99 0.98 18 0.99 0.88 1.08 0.98 0.96 0.91 19 0.97 0.94 1.11 0.94 1.03 0.93 20 0.97 1.02 1.01 1.27 0.93 0.91 21 0.95 1.01 0.95 1.00 0.99 22 0.98 0.97 0.80 0.96 1.04 23 0.88 0.92 0.94 24 0.93 1.03 0.95 25 1.01 0.97 0.77 26 0.94 1.02 27 0.92 0.98 28 1.08 0.94 29 0.88 fRatio of migratory distance (/im) in injected side to noninjected side.

i34 Appendix 3

Table 3-4. Relationship Between CNC Migratory Distance (pm) and Developmental Stage at the Time of Collection (Injected at Stage 10)

Em Stage 12 — 12* Stage 13- - 13* Stage 14 - 14* bry DM KB DM KB DM KB 0 # Non. Inj. N on/ Non. Inj. Non. Non. Inj. N on. Non. Inj. Non. Non. Inj. Non. Non. Inj. Non. Inj / / / /Inj. /Inj. Inj. Inj. Inj. 1 411 332 0.81 250 216 0.87 360 351 0.98 452 390 0.86 657 665 1.01 568 546 0.96 2 280 278 0.99 394 320 0.81 533 513 0.96 419 380 0.91 646 614 0.95 413 329 O.SO 3 324 332 1.03 224 183 0.81 579 590 1.02 573 448 0.78 644 666 1.04 399 372 0.93 4 305 298 0.98 336 327 0.97 433 423 0.98 402 382 0.95 568 534 0.94 5 272 227 0.83 278 238 0.86 464 466 1.01 384 312 0.81 6 269 253 0.94 463 451 0.97 335 326 0.97 7 276 239 0.87 461 433 0.94 453 386 0.85 8 249 232 0.93 394 383 0.97 245 239 0.98 9 268 167 0.62 535 493 0.92 256 250 0.98 10 253 239 0.94 536 552 1.03 413 392 0.95 11 236 300 1.27 494 488 0.99 428 400 0.93 12 286 238 0.83 453 434 0.96 448 395 0.88 13 249 206 0.83 347 368 1.06 14 168 153 0.91 ...... i------i------

135 Appendix 4

Figure 3-4. Effect of 46 pmoles of KB8301 on CNC Migratory Distance (pm)

Emb Injected at Stage 10- Injected at Stage 10 Injected at Stage 10+ ryo DM KB DM KB DM KB # non in,i non inJ non non in,j non inj non inj 1 230 242 251 243 402 392 348 315 508 523 460 402 2 301 295 241 221 398 381 244 224 463 482 325 317 3 295 226 356 373 485 473 343 359 529 516 391 328 4 357 356 443 361 431 446 339 293 491 507 456 402 5 390 376 235 211 481 501 411 417 564 551 466 453 6 466 416 364 387 571 584 440 388 333 343 257 292 7 258 249 365 398 180 190 501 475 505 469 343 308 8 559 572 493 421 542 537 429 400 518 507 499 443 9 518 540 331 286 487 515 442 443 395 418 641 607 10 315 354 459 374 421 385 521 495 398 447 277 293 11 468 458 383 393 451 398 326 367 330 273 517 520 12 214 207 149 172 447 459 394 384 503 485 658 602 13 528 552 278 277 387 320 522 464 501 527 352 293 14 295 290 417 442 596 572 498 433 498 467 227 285 15 571 531 472 503 439 437 205 222 430 465 474 543 16 438 457 231 211 397 351 498 433 458 492 227 285 17 451 439 280 277 486 467 375 448 527 524 564 494 18 379 357 405 396 576 589 295 308 514 474 447 405 19 476 441 493 395 353 318 455 462 462 449 466 477 20 237 240 419 397 285 336 495 507 361 318 372 313 21 345 356 492 432 362 361 513 490 451 425 457 489 22 286 287 347 337 393 389 482 464 545 505 23 210 280 357 350 481 435 24 448 452 349 326 401 446 25 366 383 439 516 476 468 26 407 387

136 Appendix 5

Figure 3-5. Effect of 92 pmoles of KB8301 on CNC Migratory Distance (/tm)

Emb Injected at Stage 10- Injected at Stage 10 Injected at Stage .0+ ryo DM DM KB- KB- DM DM KB- KB- DM DM KB- KB- # -non -inJ non inj -non -inj non inj -non -inj non inj 1 348 331 353 337 360 351 452 390 355 352 319 299 2 355 352 332 262 433 413 250 216 425 411 412 381 3 367 360 288 289 579 590 394 319 603 666 464 448 4 454 428 235 207 433 423 419 380 350 357 358 367 5 375 368 175 173 411 407 573 448 541 557 255 286 6 260 262 363 328 464 466 402 382 383 400 408 414 j 7 359 350 453 386 657 665 324 282 477 513 505 486 U~! I 1 1 is I 8 467 493 221 168 644 666 384 312 498 566 504 9 291 251 428 400 324 332 336 326 585 615 367 332 10 434 414 445 381 535 493 598 546 443 441 384 329 11 339 338 352 274 305 298 336 327 525 500 505 467 12 315 346 461 421 272 227 338 360 296 297 281 217 13 359 384 431 406 494 488 269 253 522 493 286 238 14 362 398 333 320 453 434 386 304 397 395 373 278 15 281 287 316 288 347 368 249 232 357 369 580 542 16 411 422 385 333 406 488 245 240 384 363 401 393 17 317 309 366 303 390 352 268 167 516 514 550 541 18 451 446 343 303 385 417 256 250 324 312 547 498 19 500 487 386 363 320 356 253 239 536 552 501 468 20 356 346 309 315 508 513 236 300 470 436 388 353 21 232 220 430 433 413 392 523 521 437 434 22 284 278 471 454 413 329 515 496 340 355 23 349 306 504 463 212 199 24 399 372 470 486 407 388 25 563 566 606 588 460 356 26 544 510 343 348 27 480 440 421 415 28 404 435 408 383 29 448 395

137 Appendix 6

Figure 3-6. Cross-Sectional Area of the Neural Tube in Embryos Treated with DMSO or 92 pmoles of KB8301 (p.m2)

Embryo # Injected at St. 10- Injected at St. 10 Injected at St. 10+ 1 DM KB DM KB DM KB 2 19136 14247 10738 13837 14270 10390 3 18456 16277 14144 18776 24891 17650 4 16646 14362 14169 10080 15361 9819 5 15418 15211 10377 13615 21571 20242 6 16846 11775 13152 10945 18341 i4109 7 12100 12967 19050 16324 11704 19549 8 21337 14197 19926 16078 16485 16684 9 13060 10811 17852 11869 12528 11196 10 16156 18147 16161 12986 10631 14051 11 12231 15575 17951 13399 18802 15192 12 12620 18097 17263 14664 14985 17570 13 16211 12774 12776 14083 16654 14617 14 13576 13603 19569 10965 22285 17448 15 21978 13532 19048 9734 19953 H 14738 16 19436 15923 20551 17698 18015 20653 ; 17 17022 17628 15746 19375 16437 20874 18 14484 18456 15304 18712 14224 17695 19 18437 17028 20949 10436 16644 19967 20 13220 11320 15127 18644 13980 9583 j 21 15230 19739 18453 23668 21223" 22 15660 20153 15448 19013 15230 23 16874 18158 19576 24 17695 19642 24926 25 17348 21034 18891 26 14116 23933 27 14458 23009 28 12500 24888 29 10907 15294

138 Appendix 7

Table 4-1. Effect of TIMP-2 Injection on MMP Enzymatic Activity (jnU/p.gDNA/20min)

Test Sham Ringer’s TIMP-2 1 112 115 93 2 101 97 81 3 163 158 99

139 Appendix 8

Table 4-3. Effect of TIMP-2 Injection on CNC Migration after Normalizing for Potential Difference in Embryo Sizef

Embryo # Ringer’s TIMP-2 Injected side/noninjected side Injected side/noninjected side 1 1.00 0.83 7 1.03 0.73 3 0.95 0.68 4 0.97 0.90 5 0.99 0.93 6 0.98 0.95 7 0.94 0.94 8 0.99 0.98 9 1.00 0.90 10 1.23 0.84 11 1.01 0.97 12 0.91 0.97 13 1.06 0.88 14 1.06 0.95 15 0.99 0.93 16 0.93 0.84 17 0.99 0.89 18 1.01 0.99 19 0.95 0.87 20 0.99 0.90 21 0.99 0.92 22 1.10 0.89 23 0.94 24 0.91 25 0.98 26 0.81 fRatio between migratory distance (jUm) on injected side to noninjected side.

140 Appendix 9

Figure 4-4. Effect of TIMP-2 Injection on CNC Migration (/xm)

Embryo # Ringer’s TIM!3-2 Noninjected side Injected side Noninjected side Injected side 1 464 463 479 399 2 459 472 467 340 3 573 547 411 281 4 354 342 523 470 5 589 584 510 476 6 416 406 293 279 7 341 320 546 514 8 360 357 447 439 9 481 479 422 380 10 302 373 259 218 11 222 225 363 352 12 476 434 496 479 13 486 513 517 455 14 380 403 456 432 15 378 375 549 509 16 430 401 266 224 17 412 408 368 327 18 442 445 304 303 19 291 278 418 362 20 466 460 341 306 21 540 532 508 468 22 430 474 486 432 23 420 394 24 535 489 25 473 464 26 339 275 j

141