THE ROLE of PARD3 in SCHWANN CELL DEVELOPMENT By

THE ROLE OF PARD3

IN SCHWANN CELL DEVELOPMENT

ALEXANDER JAMES BLASKY

B.S., St. Norbert College, 2004

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Cell Biology, Stem Cells and Development Program

2014

This thesis for the Doctor of Philosophy degree by

Alexander James Blasky

has been approved for the

Cell Biology, Stem Cells and Development Program

Thomas Finger, Chair

Angela Ribera

Chad Pearson

Rytis Prekeris

John Sladek

Bruce Appel, Advisor

Date 05/02/14

ii Blasky, Alexander James (Ph.D., Cell Biology, Stem Cells and Development)

The Role of Pard3 in Schwann Cell Development

Thesis directed by Professor Bruce Appel

ABSTRACT

Schwann cells are the myelin forming glial of the peripheral nervous system and are required for the rapid transmission of sensory and motor information between the central nervous system and the peripheral tissues. During development, the cells fated to become myelinating

Schwann cells undergo a stepwise sequence of events including delamination from the neuroepithelium, directed migration into peripheral tissue, axon ensheathment and synthesis of myelin membrane.

How Schwann cells regulate the transitions between these distinct behaviors is unclear. These dynamic developmental steps require cell polarity and therefore imply dynamic functions of proteins that contribute to cell polarity. However, few studies have investigated the role of any polarity proteins during the entire Schwann cell progression in vivo. In this work investigate the role of Par complex protein Pard3 in regulating polarity necessary for directed migration, axon ensheathment and myelination by Schwann cells. Time-lapse imaging revealed that neural crest delamination was normal but that migrating cells were disorganized with substantial amounts of overlapping membrane. Nevertheless, neural crest cells migrated to appropriate peripheral targets. Schwann cells wrapped motor axons and, although myelin gene expression was delayed, myelination proceeded to completion.

iii Pard3 mediates contact inhibition between neural crest cells and promotes timely myelin gene expression but is not essential for neural crest migration or myelination.

The form and content of this abstract are approved. I recommend its publication.

Approved: Bruce Appel

To Casey, for your patience, your laughter,

and your love.

v TABLE OF CONTENTS

CHAPTER

I. SCHWANN CELLS AND CELL POLARITY ...... 1

Abstract ...... 1

Schwann Cell Development ...... 2

Schwann Cell Stages ...... 2

Cell Polarity during Schwann Cell Behaviors ...... 6

Neural Crest Cells and Schwann Cell Development ...... 15

Directed Cell Migration ...... 16

Zebrafish as a Model System for Schwann Cell Development ...... 19

Schwann Cell Behaviors ...... 20

Delamination from the Neuroepithelial Tissue ...... 20

Migration to Peripheral Targets ...... 23

Schwann Cell Myelination ...... 30

Disease ...... 35

Aim and Structure ...... 35

Aim ...... 35

Structure ...... 36

II. PARD3 REGULATES CONTACT BETWEEN NEURAL CREST CELLS AND THE TIMING OF SCHWANN CELL DIFFERENTIATION BUT IS NOT ESSENTIAL FOR NEURAL CREST MIGRATION OR MYELINATION ...... 37

Abstract ...... 37

Introduction ...... 38

Results ...... 40

vi Neural Crest Delamination from the Neuroepithelium Proceeds Normally in the Absence of pard3 Function ...... 40

Pard3 Promotes Contact Mediated Inhibition but Does Not Drive Neural Crest Cell Migration ...... 46

pard3 Mutant Schwann Cells Myelinate Motor Axons ...... 53

Discussion ...... 54

Experimental Procedures ...... 62

III. PAR COMPLEX PROTEINS IN SCHWANN CELL DEVELOPMENT ...... 66

Introduction ...... 66

Results ...... 68

Schwann Cells Express mbp in Par Complex Mutants ...... 68

Motor Neuron Morphology Unchanged in Pard3 Mutants ...... 69

miR-219 and Jaw Formation in Pard3 Mutants...... 71

(PH)AKT-GF Enriched at Migratory Cell Leading Edge...... 74

Pard3-GFP is Enriched in Neuroepithelial Cell Apical Domains In Vivo...... 76

Discussion ...... 78

IV. DISCUSSION AND FUTURE DIRECTIONS ...... 81

Summary ...... 81

Future Directions ...... 87

REFERENCES ...... 90

vii LIST OF FIGURES

FIGURES

1.1. Stages of Schwann Cell Development ...... 3

1.2. Epithelial Cell Polarity Proteins during Delamination...... 8

1.3. Alignment of Zebrafish Pard3 with Known Pard3 Homologs ...... 11

1.4. Schematic Representation of Pard3 ...... 12

1.5. Comparison of Zebrafish Pard3 with Known Par3 Homologs...... 13

1.6. Directed Cell Migration...... 18

1.7. Collective Cell Migration...... 26

1.8. Contact Inhibition of Locomotion During Cell Migration...... 27

2.1. Characterization of Maternal and Zygotic pard3 Functions...... 42

2.2. Schwann Cells Exit the Dorsal Neural Tube on Schedule in pard3 Mutants...... 45

2.3. pard3 is Not Essential for Neural Crest Migration...... 47

2.4. Pard3 Mediates Contact Inhibition between Neural Crest Cells...... 50

2.5. Pard3 Localizes at Transient Points of Contact Between Neural Crest Cells...... 52

2.6. Schwann Cells Wrap Motor Axons but Delay mbp Expression in the Absence of pard3 Function...... 56

2.7. pard3 is Not Essential for Schwann Cell Myelination...... 58

3.1. Schwann Cells Delay mbp Expression in the Absence of pard3 Function...... 70

viii 3.2. Motor Neuron Specification and Branching Appear Normal in the Absence of pard3 Function...... 72

3.3. Knockdown of mir-219 Disrupts Jaw Formation...... 73

3.4 PIP3 Activation is Enriched at the Migratory Leading Edge and Sites of Cell-Cell Contact...... 75

3.5. Pard3-GFP is Localized along Neuroepithelial Cell Apical Domains In Vivo...... 77

ix CHAPTER I

SCHWANN CELLS AND CELL POLARITY

Abstract

Schwann cells are the myelin forming glial cells of the peripheral nervous system. The origin and development of the Schwann cell has been well characterized and their development has been divided into a series of maturation stages. The cells fated to become myelinating Schwann cells undergo a stepwise sequence of events including delamination from the neuroepithelium, directed migration into peripheral tissue, axon ensheathment and synthesis of myelin membrane. How Schwann cells regulate the transitions between these distinct behaviors is unclear. At each stage of their development Schwann cells are polarized, implying important roles for molecules that create cellular asymmetries. In this work we investigated the possibility that one polarity protein, Pard3, contributes to the polarized features of Schwann cells that are associated with directed migration and myelination. This chapter focuses on the molecular and morphological features of Schwann cell development, highlighting both the significant recent advances as well as the current gaps in our understanding. First, a general introduction to the maturation stages of Schwann cell differentiation and an overview of neural crest cell development and cell polarity is presented. Next, the distinct cellular behaviors of Schwann cell development are discussed in further detail.

Lastly, the significant impact of myelinating disorders on human health is discussed.

1 Schwann Cell Development

Schwann Cell Stages

Schwann cells form the myelin sheath which insulates nerve fibers and promotes the rapid conduction of action potentials in the peripheral nervous systems (Hodgkin, 1964; Jessen and Mirsky, 2005). The origin and development of the Schwann cell has been well characterized and their development has been divided into a series of maturation stages (Jessen and Mirsky, 2005; Mirsky et al., 2008; Woodhoo and Sommer, 2008) (Fig. 1.1). Schwann cells originate from multi-potent neural crest cells, which delaminate and migrate into the peripheral tissue to associate with nerve fibers (Jessen et al., 1994). Association along a nerve fiber promotes neural crest cell differentiation into Schwann cell precursors. Subsequently, Schwann cell precursors differentiate into immature

Schwann cells, and ultimately into non-myelinating and myelinating Schwann cells (Jessen and Mirsky, 2005; Woodhoo and Sommer, 2008). Although the stages have been defined, the mechanisms that regulate the transitions between these stages remains unclear. In the following sections the transcriptional and behavioral characteristics of the Schwann cell differentiation stages will be introduced, followed by a focus discussion of the Schwann cell behaviors in the latter section of this chapter.

Figure 1.1. Stages of Schwann Cell Development. Schwann cell development is divided into a series of maturation stages. Schwann cells (green) originate from multi-potent neural crest cells, which delaminate from dorsal neuroepithelium during early development. Neural crest cells migrate into peripheral tissue and differentiate into Schwann cell precursors during association with peripheral nerve fibers (blue). Schwann cell precursors further differentiate into immature Schwann cells, and ultimately into non-myelinating Remak bundles and myelinating Schwann cells.

3 Neural Crest Cells Give Rise to Schwann Cell Precursor

Neural crest cells that migrate to positions along peripheral axons give rise to Schwann cell precursors (Jessen and Mirsky, 2005). The mechanisms that control the differentiation of Schwann cell precursors from neural crest cells remain unclear. Neither the transcription factors that promote glial fate, nor the cell-extrinsic mechanisms responsible for driving glial differentiation have been clearly determined (Cheli et al., 2010; Marmigère and Ernfors, 2007; Woodhoo and Sommer, 2008). However, Schwann cell precursors can be distinguished from the neural crest cell lineage through a variety of phenotypic markers. In vivo, Schwann cell precursors can be distinguished from neural crest cells by expression of specific proteins, including Brain Fatty Acid Protein (BFAP), Desert

Hedgehog (DHH), and Protein Zero (P0) following association along an axon

(Jessen and Mirsky, 2005).

Schwann Cell Precursor Differentiate into Immature Schwann Cell

The transition from a Schwann cell precursor to an immature Schwann cell is marked by changes in Schwann cell survival responses and morphology.

Following migration, Schwann cell precursors are associated along axons and require mitotic support through axon derived neuregulin-1 type III (Nrg-1) signaling (Jessen and Mirsky, 2005; Woodhoo and Sommer, 2008). During this time, Schwann cells undergo cell proliferation or apoptotic cell death to ensure that peripheral axons are appropriately ensheathed (Jessen and Mirsky, 2005;

Lyons et al., 2005; Woodhoo et al., 2009).

4 Together, axonal derived Nrg-1 and Notch signaling drive Schwann cell precursor proliferation and differentiation to immature Schwann cell (Jessen and

Mirsky, 2005; Woodhoo et al., 2009). Inactivation of Notch signaling in Schwann cells decreases the number of Schwann cells present on an axon, indicating a role in the regulation of Schwann cell proliferation. However, this loss of Notch activity does not affect Schwann cell survival (Woodhoo et al., 2009).

The transition from Schwann cell precursor to immature Schwann cell is further defined by the initiation of axon radial sorting (Fig 1.1). Immature

Schwann cells ensheath axon bundles and begin to radially sort the ensheathed axons by extending their membrane processes into the bundle and associating along multiple small caliber axons or segregating out single large caliber axons

(Webster et al., 1973).

Immature Schwann Cells Form Non-Myelinating and Myelinating Schwann Cells

Differentiation of immature Schwann cells into non-myelinating and myelinating Schwann cells is influenced by several factors. Initially, immature

Schwann cells respond to axonal expression of Nrg-1 to form either Remak bundles or initiate myelination, with higher expression levels required for initiation of myelin formation and myelin thickness (Michailov, 2004; Taveggia et al., 2005). Additionally, signaling through G protein-coupled receptor Gpr-126 is required autonomously by Schwann cells for formation of Remak bundles and myelination, although the identity of the ligand and whether it is expressed by axons is not known (Monk and Talbot, 2009; Monk et al., 2011).

5 Immature Schwann cell differentiation into myelinating Schwann cells is further regulated by transcriptional activity (Jessen et al., 2008a). Activation of the transcription factor Sox10 (SRY-related HMG-box 10) is required throughout

Schwann cell development (Britsch et al., 2001; Finzsch et al., 2010; Jessen and

Mirsky, 2005). Sox10 is a positive regulator of the transcription factor Oct6

(octomer-binding transcription factor-6), which is also necessary for activation of the myelination program (Jaegle et al., 2003; Jaegle et al., 1996). Together, Sox10 and Oct6 form a feed-forward loop to activate the transcription factor Egr2 (early growth response-2) , also known as Krox20 (Jagalur et al., 2011; Reiprich et al.,

2010). Egr2 is essential for Schwann cell myelination. In Egr2 knock-out mice,

Schwann cells fail to wrap axons and arrest prior to myelination (Topilko et al.,

1994). The activity of Sox10, Oct6 and Egr2 leads to the suppression of myelination inhibitors, such as c-Jun and Notch, and the expression of numerous myelin genes (Jessen et al., 2008b; Jessen et al., 2008c; Srinivasan et al., 2012;

Woodhoo et al., 2009).

Cell Polarity during Schwann Cell Behaviors

The transitions in Schwann cell behavior leading to peripheral nerve myelination are associated with changes in cell polarity (Etienne-Manneville,

2008; Özçelik et al., 2010). Cell polarity is established through the asymmetric distribution of cellular organelles and proteins, and is essential for a variety of specialized cellular processes, including epithelial barrier formation, directed cell migration, and myelination (Goldstein and Macara, 2007; Özçelik et al., 2010;

Pegtel et al., 2007; Thompson, 2013). Interaction between polarity proteins

6 regulates the establishment and maintenance of cell polarity in a variety of specialized cellular behaviors.

Cell polarity is organized at the molecular level through the interaction of evolutionarily conserved polarity proteins, which can associate into multi-protein complexes (Fig. 1.2). Polarity complexes were initially identified in

Caenorhabditis elegans and Drosophila melanogaster and show a high degree of conservation throughout evolution. These are the Scribble complex, composed of

Scribble, Discs large, and Lethal giant larvae; the Crumb complex composed of

Crumbs, Pals1, and PatJ; and the Partitioning-defective (Par) complex composed of Par3, Par6, and Protein Kinase C, iota (Prkcι), also known as atypical Protein

Kinase C (aPKC) (Iden and Collard, 2008; McCaffrey and Macara, 2009;

Mellman and Nelson, 2008). Although each of these polarity complexes are known to function in a variety of specialized cellular behaviors, several recent publications have implicated members of the Par polarity complex in Schwann cell development (Chan et al., 2006; Moore et al., 2013; Tep et al., 2012).

Therefore, the following sections will highlight the Par complex and focus specifically on the Par protein Pard3.

Partitioning Defective (Par) Polarity Proteins

The partitioning defective (Par) family of polarity proteins are highly conserved in diverse animals, including worms, flies and mammals (Goldstein and Macara, 2007) (Fig. 1.3). Par family proteins were originally identified in a genetic screen in C. elegans to identify genes that regulate asymmetric zygotic division (Kemphues et al., 1988). The par genes encode six Par proteins: PAR-1

7 and PAR-4 are serine-threonine kinases, PAR-2 is a RING finger protein involved in protein ubiquitination (Levitan et al., 1994), PAR-5 is a member of the 14-3-3 protein family (Morton et al., 2002), and PAR-3 and PAR-6 are scaffolding proteins (Etemad-Moghadam et al., 1995; Hung and Kemphues, 1999).

Figure 1.2. Epithelial Cell Polarity Proteins During Delamination. Schematic representation of delamination from the neuroepithelium. A: The three polarity complexes that maintain epithelial cell apico-basolateral polarity and cell adhesion are Crumbs, Scribble, and Par. They maintain epithelial cell polarity through mutual inhibitory interactions. B: The Crumbs complex localizes to the apical most domain about tight junctions, the Par complex localizes predominantly at the tight junctions, and the Scribble complex localizes along the lateral domain.

8 1 10 20 30 40 50 60 | | | | | | | Homo sapiens ------Mus Musculus ------Xenopus laevis ------Danio rerio ------Drosophila melanogaster ------MFDVPPKCPALAN Caenorhabditis elegans MSASSTSSSSTSCPEGGEPSGSCKSSDEGESTLKKRMQQYGIASGYANSSISTLDRSQYQ

Homo sapiens ------MDPNYWIQVHRLEH Mus Musculus ------MKVTVCFGRTRVVVPCGDGRMKVFSLIQQAVTRYRKAVAKDPNYWIQVHRLEH Xenopus laevis ------MKVTVSFGRTRVVVPCGDGNLKVSSLIQQAVTRYKKAIAKDPGYWIQVHRLEH Danio rerio ------MKVTVCFGRTRVVVPCGDGNIKVQSLVQQAAMRYRRAIAKGEEYWVQVYRLEH Drosophila melanogaster KLGGLFGWRHTYKVAAKQEGIPHGQLHKSYSLTLPKRPPSPSTYSCVKPDSWVTVTHLQT Caenorhabditis elegans SLPLNGTRRVTVQFGRMKIVVPWKESDQTVGQLADAALLRYKKARGMANEDRIHVHRLEC

Homo sapiens -GDGGILDLDDILCDVADDKDRLVAVFDEQDP-----HHGGDGTS-ASSTGTQSPEIFGS Mus Musculus -GDGGILDLDDILCDVADDKDRLVAVFDEQDP-----HHGGDGTS-ASSTGTQSPEIFGS Xenopus laevis -GDGGILDLDDILCDVADDKDRLVAIYDEQDP-----HHGGDGTS-ASSTGTQSPEIFGS Danio rerio -GDGGILDLDDVLCDVADDKDRLVAVFDEQEP-----HVGGDGTS-ASSTGTQSPELYCG Drosophila melanogaster -QSG-ILDPDDCVRDVADDREQILAHFDDPGPDPGVPQGGGDGASGSSSVGTGSPDIFRD Caenorhabditis elegans ASDGGILDMDDVLEEVFDLNYDQILAITDEAN------GGSTTPTYSQIQKQQHHYAQP

Homo sapiens ELGTNN-VSAFQPYQATSEIEVTPS--VLRANMPLHVRRS-SDPALIGLS-TSVSDSNFS Mus Musculus ELGTNN-VSAFQPYQATSEIEVTPS--VLRANMPLHVRRS-SDPALTGLS-TSVSDNNFS Xenopus laevis ELGTNS-MSAFQPYQAASEIEVTPS--VLRANMPLHVRRS-SDPALVGIT-TSVSDSNFT Danio rerio EPSTSTPLSAFQPYLPHSEIEVTTS--TLRTNMPLHVRRS-SDPALLNLTAMSFSEPGSQ Drosophila melanogaster PTNTEAPTCPRDLSTPHIEVTSTTSGPMAGLGVGLMVRRS-SDPNLLASLKAEGSNKRWS Caenorhabditis elegans LPYARKFDGGPSTPIASAFGSVTVNHQAHRAASPYNVGFARSNSRDFAPQPTHSKERRDS

Homo sapiens SEEPSRKNPTRWSTTAGFLKQNTAGSPKTCDRK--KDENYRSLPRDTSNWSNQ--FQRDN Mus Musculus SEEPSRKNPTRWSTTAGFLKQNTAGSPKTCDRK--KDENYRSLPRDPSSWSNQ--FQRDN Xenopus laevis AEDPSRKNPSRWSTTAGFTKKNSSAAKGANDTK--DEE------Danio rerio PEEPSRKNPTRWSTTAGFLKPRFATGTNSLERKGRGVDTYRSLPRDAGQWSNQKEFQREK Drosophila melanogaster AAAPHYAGGD--SPERLFLDKAGGQLSPQWEED------Caenorhabditis elegans VVEVSSFDQIPQSGLRVSTPKPSRQSEDVIDGK------

Homo sapiens ARSSLSASHPMVGKWLEKQEQDEDGTEEDNSRVEPVG-HADTGLEHIPNFSLDDMVKLVE Mus Musculus ARSSLSASHPMVDRWLEKQEQDEEGTEEDSSRVEPVG-HADTGLENMPNFSLDDMVKLVQ Xenopus laevis ------EAEENSRVEPVG-HADTSLERTSSFSLDDMVKLVE Danio rerio ARSSLSANHPMVDRWLERQEQ----DEEENGRIEPVG-RADTCLEHMGVRSLDDIVKLVE Drosophila melanogaster ----DDPSHQLKEQLLHQQQPHAANGGSSSGNHQPFARSGRLSMQFLGDGNGYKWMEAAE Caenorhabditis elegans ------PMNQPILRSSLRTEASGSRTEEATPVKQSRVTLSPEVEKKLAEQDERKSER

Homo sapiens VPNDGGPLGIHVVPFSARGGRTLGLLVKRLEKGGKAEHENLFREND---CIVRINDGDLR Mus Musculus VPNDGGPLGIHVVPFSARGGRTLGLLVKRLEKGGKAEQENLFHEND---CIVRINDGDLR Xenopus laevis VSNDGGPLGIHVVPYSARGGRTLGLLVKRLEKGGKAERENLFHEND---CIVRINNGDLR Danio rerio VSNDGGPLGIHVVPFSGRDRRTLGLLVKRLERGGKADVQGLFQEND---CIIRINNGDLR Drosophila melanogaster KLQNQPPAQQTYQQGSHHAGHGQNGAYSSKSLPRESKRKEPLGQAY---ESIREKDGEML Caenorhabditis elegans RKHYDKNPGRFARGSDRKSRITDALLDARDRIADQLESQNPAEETKSQMIRVKIDQGPMP

Homo sapiens NRRFEQAQHMFRQAMRTP----IIWFHVVPAANKEQYEQLSQSEKNNYY----SSRFSPD Mus Musculus NRRFEQAQHMFRQAMRAR----VIWFHVVPAANKEQYEQLSQREKNNYS----PGRFSPD Xenopus laevis NRRFEQAQNMFRQAMRSP----VIWFHVVPAANKEPYEQLSQSENNSYY----SNQHLHD Danio rerio NVRFEQAQNMFRQAMRSP----VILFHVVPTSMRSQYEQISHNEHNPRANMDLSGRFSPD Drosophila melanogaster LIINEYGSPLGLTALPDKEHGGGLLVQHVEPGSRAERGRLRRDDRILEIN---GIKLIGL Caenorhabditis elegans GTSLVTFPPIPEKSENEKQLGIEVNAVFDESSELPGTSEPTKLSSVQIMKIEDGGRIAKD

Homo sapiens SQYID-NRSVNSAGL-HTVQRAPRLNHPPEQIDSHSRLPHSAHPSGKPPSAPASAPQNVF Mus Musculus SHCVA-NRSVANNAP-QALPRAPRLSQPPEQLDAHPRLPHSAHASTKPPAAPALAPPSVL Xenopus laevis SQYMD-SRNFPSTGPDHTAQRLPRQSSQADPLDSYSHLPQSINSAGKPPTGLTPSPQRAV Danio rerio SLTND-LDSAAHRLAQHRPQ------PPNNHLDTGSPVHHLVGSSGKPPTGHTSSPQRGL Drosophila melanogaster TESQV-QEQLRRALESSELRVRVLRGDRNRRQQRDSKVAEMVEVATVSP---TRKPHAAP Caenorhabditis elegans GRIRVGDCIVAIDGKPVDQMSIIRVRASISDLAAVTSRPVTLIINRSLESFLEQESSAKP

Homo sapiens STTVSSGYNTKKIGKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVKNILPRGAAIQDG Mus Musculus STNVGSVYNTKKVGKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVKNILPRGAAIQDG Xenopus laevis NSPTNSGYATKK-GKKFYIQLKKGVEGLGFSITSRDVPLGGSAPIYVKNILPRGAAIQDG Danio rerio SPAPTTGF-TKKVGRRLGIQLKKGPEGLGFSITSRDVPLGGSAPIYVKNILPRGAAIQDG Drosophila melanogaster VGTSLQVANTRKLGRKIEIMLKKGPNGLGFSVTTRDNPAGGHCPIYIKNILPRGAAIEDG Caenorhabditis elegans IQSALQQANTQYIGHTTVVELIKSSNGFGFTVTGRETAKG-ERLFYIGTVKPYGVALG-- Figure 1.3. Alignment of zebrafish Pard3 with known Pard3 homologs

9 Homo sapiens RLKAGDRLIEVNGVDLVGKSQEEVVSLLRSTKMEGTVSLLVFRQEDAFHPRELNAEPSQM Mus Musculus RLKAGDRLIEVNGVDLAGKSQEEVVSLLRSTKMEGTVSLLVFRQEEAFHPREMNAEPSQM Xenopus laevis RMKAGDRLIEVNGVDLTGRTQEEVVSLLRSTKMDGAVNLLVLRQEDSFHPCELSPEPSQV Danio rerio RLKAGDRLLEVNGVDLNGRGQEEVVSLLRATPMGGTVTLVVLRQEETFIPREMNAEP-AI Drosophila melanogaster RLKPGDRLLEVDGTPMTGKTQTDVVAILRGMPAGATVRIVVSRQQELAEQADQPAEKSAG Caenorhabditis elegans HLKSGDRLLEINGTPTGQWTQSEIVEKLKETMVGEKIKFLVSRVSQSAIMSTSASSENKE

Homo sapiens QIPKETKAEDEDIVLTPDG------Mus Musculus QTPKETKAEDEDVVLTPDG------Xenopus laevis PNARESKTEEEELVLTPDG------Danio rerio QNTREWKVEEEELVLTPDG------Drosophila melanogaster VAVAPSVAPPAVPAAAAPAPPIPVQKSSSARSLFTHQQQSQLNESQHFIDAGSESAASND Caenorhabditis elegans NEETLKVVEEEKIPQKLPLPALMTPPVPKDTPALSPSG------

Homo sapiens ------TREFLTFEVPLNDSGSAGLGVSVKGNRSK------ENHA Mus Musculus ------TREFLTFEVPLNDSGSAGLGVSVKGNRSK------ENHA Xenopus laevis ------TREFLTFEIPLNDSGSAGLGVSVKGNRSK------ENHA Danio rerio ------TREFLTLEVPLNDSGSAGLGVSVKGNRSK------ESHA Drosophila melanogaster SLPPSSNSWHSREELTLHIPVHDTEKAGLGVSVKGKTCSNLNASGSSASSGSNGLMKHDG Caenorhabditis elegans ------ASRFEIVIPFINGSSSAGLGVSLKARVSKKSN------GSKV

Homo sapiens DLGIFVKSIINGGAASKDGRLRVNDQLIAVNGESLLGKTNQDAMETLRRSMS-TEGNKRG Mus Musculus DLGIFVKSIINGGAASKDGRLRVNDQLIAVNGESLLGKANQEAMETLRRSMS-TEGNKRG Xenopus laevis DLGIFVKSIINGGAASKDGRLHINDQLVAVNGESLLGKTNQDAMETLRKSMS-TEGNKRG Danio rerio DLGIFVKSIINGGAACKDGRLRVNDQLIAVNGESLLGKTNQDAMETLRKSMS-TEGNKRG Drosophila melanogaster DLGIFVKNVIHGGAASRDGRLRMNDQLLSVNGVSLRGQNNAEAMETLRRAMVNTPGKHPG Caenorhabditis elegans DCGIFIKNVMHGGAAFKEGGLRVDDRIVGVEDIDLEPLDNREAQAALAKKLK-----EVG

Homo sapiens MIQLIVARRISKCN------ELKSPGSPPGPELP Mus Musculus MIQLIVARRISRCN------ELRSPGSPAAPELP Xenopus laevis MIQLIVARRVKLS------ELESPGTPSGPELP Danio rerio MIQLIVARRINKRL------EGESRSSPRGLERT Drosophila melanogaster TITLLVGRKILRSASSSDILDHSNSHSHSHSNSSGGSNSNGSGNNNNSSSNASDNSGATV Caenorhabditis elegans MISSNVRLTISRYN------ECNPGQISRDLSR

Homo sapiens IETALDDRERRISHSLYSGIEGLDESPSRNAALS-RIMGKYQLSPTVN------MPQD Mus Musculus IETELDDRERRISHSLYSGIEGLDESPTRNAALS-RIMGKCQLSPTVN------MPHD Xenopus laevis VETMLDDRERRISHSLYSSIEGFDESPTRNAALN-RIMGKYQLSPTVN------MPQD Danio rerio LSPSPDDHERRISHSLYG-IEGLDDNLRPQASTNNRKMNHYQLSPTVN------MPQD Drosophila melanogaster IYLSPEKREQRCNGGGGGGSAGNEMNRWSNPVLDRLTGGICSSNSAQPSSQQSHQQQPHP Caenorhabditis elegans ITVDASSPSPSSRMSSHTAPDSLLPSPATRGTSSSGADSSHSRQSSAS------SAVP

Homo sapiens DTVIIEDDRLPVLPPHLSDQSSS------SSHDDVGFVTADAGTWAK Mus Musculus DTVMIEDDRLPVLPPHLSDQSSS------SSHDDVGFIMTEAGTWAK Xenopus laevis DTVIIEDDRTPVLPSQLSDHSSS------SSHDDVGCVDSTS-AWSK Danio rerio DTVIIEDDRPHVLPIQLSDQSSS------SSHDDMGFAVETP--PPP Drosophila melanogaster SQQQQQQRRLPAAPVCSSAALRNESYYMATNDNWSPAQMHLMTAHGNTALLIEDDAEPMS Caenorhabditis elegans AVPARLTERDSIVSDGTSRNDES------ELPDSADPFNREG--LGR

Homo sapiens AAISDSADCSLSPDVDPVLAFQREGFGRQ------SMSEKRTKQFSDASQLDFVK Mus Musculus ATISDSADCSLSPDVDPVLAFQREGFGRQ------SMSEKRTKQFSDASQLDFVK Xenopus laevis TVASESTESTLSPDVDPSLAFQREGFARQ------SMSEKRTKQFGDASQLDFVK Danio rerio PWEPELPDSSSSANAEG--QFQREGFGRQ------SMSEKRTKQYGDAGQLDIIK Drosophila melanogaster PTLPARPHDGQHCNTSSANPSQNLAVGNQGPPINTVPGTPSTSSNFDATYSSQLSLETNS Caenorhabditis elegans KSLSEKRGMGAAADPQHIKLFQDIKHQRQ------NSAPTSSTQKRSKSQPRSSS

Homo sapiens TRKSKSMDLG---IADETKLNTVDDQKAGSPSRD------VGPSLGLKKSSSLE Mus Musculus TRKSKSMDL----VADETKLNTVDDQRAGSPSRD------VGPSLGLKKSSSLE Xenopus laevis TRKSKSMDL----VADETKISAMAGQNSGSPSRD------VGPSLGLKKSSSLE Danio rerio TRKSKSMDL----VADEINLTQCTENHTGSSTRD------VGPSLGLKKSSSLE Drosophila melanogaster GVEHFSRDALGRRSISEKHHAALDARETGTYQRNKKLREERERERRIQLTKSAVYGGSIE Caenorhabditis elegans QRNYRSPMK-----LVDLPTTAAASASTNSQNLD------DSDMLNRRSQSME

Homo sapiens SLQTAVAEVTLNGDIPFHRPRPRIIRGRGCNESFRAAIDKSYDKPAVDDDDE-GMET--- Mus Musculus SLQTAVAEVTLNGNIPFHRPRPRIIRGRGCNESFRAAIDKSYDKPMVDDDDE-GMET--- Xenopus laevis SLQTAVAEVTLNGDIPFHRPRPRIIRGRGCNESFRAAIDKSYDKPSADDDDE-GMETCTN Danio rerio SLQTAVAEVTLNGDMPFHRPRPRIIRGRGCNESFRAAIDKSYDRPAANEDEEECMDT--- Drosophila melanogaster SLTARIASANAQFSGYKHAKTASSIEQRETQQQLAAAEAEARDQLGDLGPSLGMKKSSSL Caenorhabditis elegans SINRPVESILRGTGQIPTGSSSKVQFMQAASPDQHPFPPGAALLRLKNEESRSRDKS--- Figure 1.3. Alignment of zebrafish Pard3 with known Pard3 homologs

Homo sapiens ------LEEDTEESSRSGRES------VSTASDQP--SHSLERQMNGNQEKG Mus Musculus ------LEEDTEESSRSGRES------VSTSSDQP--SYSLERQMNGDPEKR Xenopus laevis KSELEPINSSSEWVEEDTEESSKSGRES------VSTASDQH--SHSLERQVNGSSQ-- Danio rerio ------LEEDTEGSSRSGRDS------VSTVADLTPLPVTEQQLINGNQP-- Drosophila melanogaster ESLQTMVQELQMSDEPRGHQALRAPRGRGREDSLRAAVVSEPDASKPRKTWLLEDGDHEG Caenorhabditis elegans ------RRKSMGNAMRNFFGFGS-----KSRDASPEKTPTESVQLRSVERPKS

Homo sapiens DKTDRKKDKTGKEKKKDRDKEKDKMKAKKGMLKGLGDMFRFGKHRKDDKIEKTGKIKIQE Mus Musculus DKTERKKDKAGKDKKKDREKEKDKLKAKKGMLKGLGDMFRFGKHRKDDKMEKMGRIKIQD Xenopus laevis EKSDRKKDKSGKEKKKDRDKDKNKAK-KGGMLKGLGDMF------Danio rerio -ENDKKKEKGGKDKKK---PEKEKGKTKKGMLKGLGEMFRFGKYRKDERLDG-AKWKAEE Drosophila melanogaster GFASQRNGPFQSSLNDGKHGCKSSRAKKPSILRGIGHMFRFGKNRKDGVVPVDNYAVNIS Caenorhabditis elegans IIDERNNGSSERAPPPLPPHQSQRRGSGGNVFVDYGEPYGLIPQYPHNTTSG------

Homo sapiens SFTSEEERIRMKQEQERIQAKTREFRERQ-ARERDYAEIQDFHR---TFGCDDELMYGGV Mus Musculus SFTSEEDRVRMKEEQERIQAKTREFRERQ-ARERDYAEIQDFHR---TFGCDDELLYGGM Xenopus laevis ------KIQAKTRELRERQ-AREKDYAEIQDYAK---THGSLDESPYAGA Danio rerio THASEEETRRMKQEQERIQAKTREIRQRQ-ARERDYAEIQDFSRSTLTSLPPEEPPYAGI Drosophila melanogaster PPTSVVSTATSPQLQQQQQQQLQQHQQQQQIPTAALAALERNGKPPAYQPPPPLPAPNGV Caenorhabditis elegans -----YESYADSELYDRYAAHRYHPRGGPIIDEDEYIYRQQSTS---GNSPINTSSYVNY

Homo sapiens SSYEGS-----MALNARPQSPREGHMMDALYAQVKKPRNSKPSPVDSNRSTPSNHDRIQR Mus Musculus SSYEGC-----LALNARPQSPREGHLMDTLYAQVKKPRSSKPG--DSNRSTPSNHDRIQR Xenopus laevis GLYDTCSNSGTLHLNNRPQSPRDGKQTEALYAQVKKPRSSKPSPVDR------Danio rerio GSLEHG------GYHRIHTPP-----DSPYTQKQNGRNGHPSTSDRY------Drosophila melanogaster GSNGIHQN---DIFNHRYQHYANYEDLHQQHQQHQISGGDSTTSISETLSESTLECMRQQ Caenorhabditis elegans GLPASN------AYHVGSRIPPQTSSGSISKTSGAMRRVYPAEYDEDVAYHQQIPQQST

Homo sapiens LRQEFQQAKQDEDVEDRRRTYSFEQPWPNARPATQSGRHSVSVEVQMQRQRQEERESSQQ Mus Musculus LRQEFQQAKQDEDVEDRRRTYSFEQSWSSSRPASQSGRHSVSVEVQVQRQRQEERESFQQ Xenopus laevis ------Danio rerio ------Drosophila melanogaster VIRQRIKVEAESRRHQHYHSQRSARSQDVSMHSTSSGSQPGSLAQPQAQSNGVRPMSSYY Caenorhabditis elegans RYQQGSGSGRGNADYHHMFNSWFAYTGGGAVGAAPVIKSSYGSSPVRIAAASAIERGESF

Homo sapiens AQRQYSSLPRQSRKNASSVSQDSWEQNYSPGEGFQSAKENPRYSSYQGSRNGYLGGHGFN Mus Musculus AQRQYSSLPRQSRKNASSISQDSWEQNYAPGEGFQSAKENPRYSSYQGSRNGYLGGHGFN Xenopus laevis ------Danio rerio ------Drosophila melanogaster EYETVQQQRVGSIKHSHSSSATSSSSSPINVPHWKAAAMNGYSPASLNSSARSRGPFVTQ Caenorhabditis elegans VVEPVSGSSASATDRRGRSTSSGAVASGSSSTGFQYAAKEKYADARSGKFNGGSTRLFIP

Homo sapiens ARVMLETQELLRQEQRRKEQQMKKQPPSEGPSNYDSYKKVQDPSYAPPKGPFRQDVPPSP Mus Musculus ARVMLETQELLRQEQRRKEQQLKKQPPADG------VRGPFRQDVPPSP Xenopus laevis ------Danio rerio ------Drosophila melanogaster VTIREQSSGGIPAHLLQQHQQQQLQQ------QQPTYQTV Caenorhabditis elegans RHGGGLSAAAFATNFGG------EAYETRGG

Homo sapiens SQVARLNRLQTPEKGRPFYS--- Gray = CR1 domain Mus Musculus SQVARLNRLQTPEKGRPFYS--- Orange = PDZ1 Xenopus laevis ------Blue = PDZ2 Danio rerio ------Green = PDZ3 Drosophila melanogaster QKMSGPSQYGSAAGSQPHASKV- Yellow = PKC binding region Caenorhabditis elegans GAGGSPSQYRRRDQGPPHRFPQY Red = Lesion (Y203*)

Figure 1.3. Alignment of zebrafish Pard3 with known Pard3 homologs. Zebrafish Pard3 was aligned with Pard3 homologs from H. sapiens, M. Musculus, X. laevis, D. melanogaster and C. elegans. The N-terminal conserved CR1 domain is highlighted in Grey. The PDZ1 domain is highlighted in Orange, the PDZ2 domain is highlighted in Blue and the PDZ3 domain is highlighted in Green. The aPKC binding region (Yellow) is also conserved. The pard3fh305 lesion site is highlighted in Red. This alignment was prepared using the ClustalW method with Genious software.

Figure 1.4. Schematic representation of Pard3.. Pard3 has a conserved oligomerzation domain (CR-1), three PDZ domains (PDZ1-3) and a Prkcı binding domain. The pard3fh305 lesion, which changes a tyrosine to a stop codon at amino acid position 203 (Y203*) occurs after the oligomerization domain and before the PDZ binding domains (red line).

12 Pard3 is a highly conserved scaffold protein which contains an amino- terminal oligomerization domain, three PSD-95/Discs-large/ZO-1 (PDZ) domains and a carboxyl-terminal Prkcι-interaction domain (Fig. 1.4). When compared to Pard3 homologs in multiple species, the zebrafish Pard3 amino acid sequence is highly conserved, particularly among vertebrates. Zebrafish Pard3 has 65.6% identity with Homo sapiens, 64.7 % identity with Mus musculus, and

47.2% identity with Xenopus laevis (Fig. 1.5).

The functional domains of the Pard3 homologs show an even higher degree of conservation. The CR-1 domain has 78% identity between zebrafish and human. Zebrafish Pard3 contains three PDZ domains, which are also highly conserved, sharing 81%, 83%, and 94% identity respectively, with the three human PDZ domains. The carboxyl-terminal Prkcı-interaction domain is 100% identical to the human domain.

Figure 1.5. Comparison of zebrafish Pard3 with known Par3 homologs. The percent identity and percent similarity between zebrafish Par3 and known Par3 homologs. These data were generated by ClustalW alignment using Geneious software.

The high level of conservation among diverse species is consistent with the idea Pard3 acts as a signaling hub, mediating cell polarity and cytoskeletal

13 regulation through binding of its functional domains. The amino-terminal domain of Pard3 (conserved region, CR-1) functions as an oligomerization domain, providing a molecular mechanism for localized enrichments of the Par complex within the cell (Benton and St Johnston, 2003; Feng et al., 2007;

Mizuno et al., 2003). The CR-1 domain is essential for cortical localization of polarity proteins during C. elegans zygotic asymmetric division, although recent evidence suggests this domain may be non-essential for later development (Li et al., 2010). The CR-1 domain also functions in cytoskeletal organization. The

Pard3 CR-1 domain binds, bundles and stabilizes microtubules through an intramolecular regulatory mechanism. Further, the expression of Pard3 CR-1 domain alone is sufficient to polarize neurons through the binding and stabilization of microtubules (Chen et al., 2013).

Pard3 also interacts with multiple proteins through binding along its PDZ domains. The first PDZ domain within Pard3 binds the Par complex partner

Pard6 and interacts with cell cytoskeletal regulator proteins junctional adhesion molecule (Jam) and Nectin (Ebnet et al., 2003; Joberty et al., 2000; Lin et al.,

2000; Suzuki and Ohno, 2006; Takekuni et al., 2003). The second and third PDZ domains interact with phosphoinositide phosphates (PIPs) and the PI(3,4,5)P3 phosphatase PTEN (Feng et al., 2008; Wu et al., 2007). Pard3 PDZ domains 2 and 3 also bind microtubules in a conformational dependent manner (Chen et al.,

2013).

Pard3 regulates the activity of several proteins through interactions along its carboxyl-terminal domain. Pard3 interacts with Prkcı through a high conserved carboxy-terminal binding domain, which when bound inhibits Prkcı

14 kinase activity (Izumi et al., 1998; Nagai-Tamai et al., 2002). Further, Pard3 interacts with Tiam1 (T lymphoma invasion and metastasis), a Rac1 GTPase- specific GTP exchange factor, to inhibit its exchange activity. This interaction provides a direct connection between Pard3 and Rac activity, which is a main mediator of cytoskeletal organization (Chen and Macara, 2005; Pegtel et al.,

2007).

Neural Crest Cells and Schwann Cell Development

Schwann cells located along peripheral nerves originate from multi-potent neural crest cells. Neural crest cells originate along the neural plate at the border between the neural and non-neural ectoderm early in development (Bronner-

Fraser and LeDouarin, 2012). As the neural plate folds inward to form the neural tube neural crest cells are deposited along the dorsal neural tube underlying the non-neural ectoderm. Neural crest cells delaminate from the neuroepithelial tissue along the dorsal neural tube, which involves an epithelial-to-mesenchymal transition (EMT) (Duband et al., 1995; Strobl-Mazzulla and Bronner-Fraser,

2012). The timing of delamination of neural crest cells can vary along the body axis and involves a variety of cellular mechanisms, including down regulation of cellular adhesions and disruption of epithelial cell apico-basolateral polarity.

(Theveneau and Mayor, 2012). Following delamination, neural crest cells migrate extensively throughout the developing embryo guided by multiple extracellular cues that influence migratory paths. Previously, neural crest cells were thought to migrate individually and with limited cell-cell contact. However, recent advances in cell tracking and time-lapse microscopy have provided insight into this

15 migratory behavior and show neural crest cells exhibit collective cell migration, wherein cell-cell contact influences migratory behaviors (Carmona-Fontaine et al., 2008; Matthews et al., 2008; McLennan et al., 2012; Teddy and Kulesa,

2004). These interactions promote neural crest cell migration within specific migratory paths where they eventually stop and differentiate into a vast array of different tissues and cell types, including Schwann cells (Jessen and Mirsky,

2005; Le Douarin and Kalcheim, 1999). To what extent cell-cell contact influences neural crest cell migration remains unclear.

Directed Cell Migration

Cell migration is essential throughout the life of a multi-cellular organisms, from the early events of tissue morphogenesis and neurogenesis to immune functions and wound healing. The general principles of cell migration are fairly well understood and are discussed in this section.

Directed cell migration requires generation of leading and trailing domains within the cell (Fig. 1.6). At the leading edge, directionally migrating cells extend membrane processes through dynamic cytoskeletal activity.

Membrane extensions can either be lamellipodia, which are flat, broad membrane protrusions, or filopodia, which are thin actin-rich membrane extensions involved in environment probing (Andrew and Insall, 2007;

Arrieumerlou and Meyer, 2005; Petrie et al., 2009). These membrane extensions will form dynamic attachments to the surrounding substrate. These attachments can be quickly disassembled or stabilized to generate directional movement depending on the collective cellular response. Following attachment of the

16 leading membrane to the surrounding substrate the cell can generate traction and contractile forces to migrate. The cell-substrate connection is mediated through the interaction of cytoskeletal proteins and adhesion molecules such as cadherins and integrins that directly bind the substrate molecules (Huttenlocher and

Horwitz, 2011; McKeown et al., 2013).Traction forces at the leading edge generate cell displacement in the direction of attachment, while the contraction force initiate movement of the rear of the cell (Petrie et al., 2009; Ridley, 2011). The rear of the cell is also attached to the surrounding substratum and the adhesions must be disassembled in order to contract the rear of the cell.

Rac1, Cdc42, and RhoA have more specific functions during directed cell migration. Activated Rac1 mediates membrane protrusions along the leading edge of migrating cells through the regulation of actin dynamics (Nobes and Hall,

1995; Wennerberg and Der, 2004). The position of the centrosome, which serves as the microtubule-organizing center (MTOC), in front of the cell nucleus is a strong indicator of cell polarity and direction of migration (Dupin and Etienne-

Manneville, 2011). Cdc42 plays an integral role in defining the orientation of cell migration in chemotaxis and wound-induced migration through centrosome localization and regulation of actin dynamics within filopodia (Etienne-

Manneville, 2005; Etienne-Manneville and Hall, 2001; Gomes et al., 2005;

Tapon and Hall, 1997). Cdc42 has been shown to function at the leading edge during cell migration (Etienne-Manneville, 2005; Huo et al., 2011).

Figure 1.6. Directed Cell Migration. Directional migration is the result of membrane process extension at the leading edge and membrane retraction at the trailing edge. A: Directional migration requires polarized localization of Rho GTPase family members for regulation of cytoskeletal dynamics. B: Neural crest cell exhibiting directed migration in vivo.

18 Although the general principles of directed cell migration in individual cells have been described, our understanding of the complex regulation of these principles during collective cell migration is limited. In Chapter II, the role of cell polarity protein Pard3 as a possible mediator of directed cell migration during collective cell migration is discussed.

Zebrafish as a Model System for Schwann Cell Development

Zebrafish are an excellent model system to study vertebrate nervous system development and human diseases. The popularity of the zebrafish model can be attributed to several key features. First, zebrafish embryos are optically transparent, which allows researchers to observe the processes of embryonic development in vitro. Combining the use of fluorescent markers and the cutting edge microscopy, one can track and trace certain cells in zebrafish embryos to study cell/tissue-specific gene expression, cell differentiation, and cell behaviors as well. Further, genetic modification can more easliy be addressed in zebrafish compared to other vertebrate organisms, such as the mouse, and rat. Nowadays, transgenic zebrafish lines are commonly used in scientific research. Extensive use of high-throughput genetic screenings provides a large amount of information of human disease-relation genes and mutations. Gaining novel insight of the function of these genes and mutations could be extremely time-consuming without a proper model system. In addition, zebrafish have a rapid external development and high fecundity which allow researchers to take advantage of using zebrafish as a model system.

19 Schwann Cell Behaviors

Delamination from the Neuroepithelial Tissue

Neural crest cell delamination from the dorsal neuroepithelium is the initial transition in cellular behavior during Schwann cell development. Neural crest cells delaminate from the relatively static neuroepithelial tissue and transform into a highly migratory cell type, a process known as epithelial to mesenchymal transition (EMT). EMT involves loss of cellular adhesions and disruption of cell polarity mediated through the activation of complex gene regulatory networks (Sauka-Spengler and Bronner-Fraser, 2008)

Neural crest cell delamination from the neuroepithelial tissue involves the orchestrated activity of signaling pathways and transcription factors. Neural crest cells arise at the border of neural and non-neural ectoderm around the time of neural tube closure in response to a combination of signaling factors, including

FGF, WNT, and BMP (Sauka-Spengler and Bronner-Fraser, 2008). These factors promote neural crest cell specification through the activation of several key transcription factors. The activity of transcription factors Snail, Twist and Zeb drive the changes in gene expression necessary for neural crest cell delamination and EMT. These transcription factors are activated early in EMT and control expression of each other, as well as cooperate during target gene regulation

(Peinado et al., 2007). Snail, Twist and Zeb down-regulate cytoskeletal proteins, including E-cadherin, claudins and occludins, and cell polarity proteins Crumbs,

PatJ and Pals1 (Lamouille et al., 2014).

20 Coincident with changes in transcriptional regulation, neural crest cells alter their cellular morphology and begin to delaminate from the surrounding neuroepithelial tissue. Neural crest delamination can occur via several processes including asymmetric division, force generation, and down-regulation of cellular adhesion complexes and often occurs as a result of multiple factors (Ahlstrom and Erickson, 2009; Berndt et al., 2008; Clay and Halloran, 2010; Theveneau and Mayor, 2012). The dynamic reorganization of cellular adhesions is a key characteristic of neural crest cell delamination. As neural crest cells originate from the neuroepithelial tissue they initially exhibit epithelial cell adhesion, which consists of tight junctions and adherens junctions. Tight junctions are comprised of two main transmembrane proteins, Claudins and Occludins. Prior to delamination both Claudins and Occludins are down-regulated, including through direct transcriptional repression by Snail (Aaku-Saraste et al., 1996;

Ikenouchi, 2003; Martínez-Estrada et al., 2006). Further evidence in support of cell adhesion as a regulator of neural crest delamination was shown by the inhibition of the tight junction protein Cingulin which resulted in an increase in the size of the migratory neural crest domain (Wu et al., 2011).

Regulation of adherens junctions has also been implicated in neural crest cell delamination. Adherens junctions are formed by several proteins, including

Cadherins and Catenins. Cadherins are transmembrane proteins that stabilize cell-cell adhesions through the homophilic interaction of their extracellular domains (Taneyhill, 2008). These cell-cell adhesion are also anchored to the actin or microtubule cytoskeleton through interaction with adherens junction- associated Catenins (Etienne-Manneville, 2011). The process of altering the

21 expression of Cadherins family members located on the cell surface, known as

Cadherin switching, from epithelial associated to mesenchymal associated

Cadherins promotes the delamination of neural crest cells and initially defines the pre-migratory neural crest cells in mouse, chick, and zebrafish (Chu et al.,

2006; Inoue et al., 1997; Liu et al., 2008; Nakagawa and Takeichi, 1995;

Wheelock et al., 2008). However, instances of neural crest cell delamination in the absence of complete down-regulation of cellular adhesions have been described and occur as a result of force generated through cytoskeletal reorganization (Ahlstrom and Erickson, 2009).

Increasing evidence implicates disruption of polarity proteins in neural crest cell delamination and EMT. Snail and Zeb1, known transcriptional mediators of neural crest EMT, directly repress the transcriptional activity of the polarity protein Crumbs (Aigner et al., 2007; Karp et al., 2008; Spaderna et al.,

2008; Whiteman et al., 2008). As discussed previously, neural crest cell delamination from the neuroepithelium is accompanied by disassembly of cellular adhesions such as tight junctions (Aaku-Saraste et al., 1996; Hay, 1995;

Sauka-Spengler and Bronner-Fraser, 2008). Pard3 is required for the establishment and maintenance of epithelial cell adhesions. Pard3 localizes to tight junctions at the apical-lateral boundary of epithelia (Izumi et al., 1998) where, together with junctional adhesion molecule (Jam) and Nectin, it promotes tight junction assembly and stability (Afonso and Henrique, 2006; Chen and

Macara, 2005; Ebnet et al., 2001; Ooshio et al., 2007). Disruption of polarity proteins has been implicated in EMT and increased cancer metastasis

(Muthuswamy and Xue, 2012). Recently, the loss of Pard3 has been shown to

22 promote cancer invasiveness and metastasis due to loss of cell-cell cohesion

(McCaffrey et al., 2012; Xue et al., 2013). Despite the known role of Pard3 in regulation of epithelial cell polarity and adhesions, little is known about Pard3 function during neural crest cell delamination. In Chapter II, loss of Pard3 function during neural crest cell delamination is discussed.

Migration to Peripheral Targets

Over the past three decades many of the molecular mechanisms that regulate individual cell migration have been investigated and characterized. Yet, during the development of multicellular organisms often cells do not migrate as single isolated entities, but rather interact with neighboring cells and migrate collectively. This idea has lead to the emerging field of collective cell migration and raised questions about the function of cell-cell contact and underlying mechanisms that control these interactions during migration. In contrast to individual cell migration, the mechanics of collective cell migration have only recently been the focus of investigation (Reffay et al., 2014; Reffay et al., 2011).

How cell-cell interactions are involved in directing cell migration in vivo and the mechanisms that mediate response during cell-cell interaction are not fully understood.

Collective cell migration occurs in a variety of context in vivo, including normal morphogenetic events during development and pathological manifestations, such as cancer metastasis (Friedl and Gilmour, 2009). Collective cell migration can occur in a variety of distinct ways. Collectively migrating cells have been characterized as migratory sheets during cranial development

23 (Carmona-Fontaine et al., 2008), as clusters during zebrafish lateral line formation (Haas and Gilmour, 2006), and also as streams of cells or in chains during neural crest cell migration (Carmona-Fontaine et al., 2008; Teddy and

Kulesa, 2004) (Fig 1.7).

Contact Inhibition of Locomotion

Contact inhibition of locomotion (CIL) is a term used to describe the outcome of the collision of two migratory cells, where the cells involved change migration direction after contacting another cell (Fig. 1.8). This event involves the retraction of the leading edge following contact. Advances in the field of cell migration, including identification of molecular mechanisms and improved microscopy techniques, lead to the generation of cell migration models focused on cell polarization and chemotaxis. However, these models neglected the influence of CIL during collective migration. Recent work using the neural crest lineage as a model has suggested a prominent role for CIL in collective cell migration.

Two main issue regarding CIL in neural crest cell migration remain unclear and are addressed in this thesis: How does CIL regulate neural crest cell migration in the context of multiple different migratory effectors, and what are the molecular mediators of CIL in neural crest cells?

Neural Crest Cell Migration

Neural Crest Cells are an excellent model to study the influence of polarity on migratory mode. NCC have been observed to display both streaming and chain

24 migration in vivo. Neural crest cells are accessible to in vivo observation and intervention in zebrafish, and other vertebrate model systems. Neural Crest cell arise from the same population of cell, however they exhibit different migratory modes depending on the local tissue environment. The different tissue environment neural crest migrate through are comprised of different permissive and restrictive cues, including ECM, nascent tissues and secreted molecules.

Comparison of neural crest cell migration in multiple animal models reveals a high degree of conservation in the cellular behaviors and regulatory mechanisms involved (Theveneau and Mayor, 2012). Neural crest cells are divided into several distinct subpopulations based on their tissue location and during their migration they display differing migratory strategies and behaviors.

Cranial neural crest cells give rise to cranial structures including the cranial ganglion and cartilage and bone structures in the face and neck. Cranial neural crest cells migrate way from the neuroepithelium as collective sheets of cells. This migration behavior appears well conserved among model organisms

(Theveneau and Mayor, 2011). Cranial neural crest cell migration is controlled by positive and negative external cues. As they migrate cranial crest cell respond to positive cues from the external environment, which include VEGF, FGF, PDGF, and SDF1, also known as CXCl12 and permissive cues from the extracellular matrix.

Figure 1.7. Modes of Collective Cell Migration. A. Collectively migrating cell exhibit various cell-cell interactions. Sheet migration involves a tightly associated cohort of cells that exhibit robust cell-cell adhesions. Membrane process extension is limited by cell contact and is restricted to lead cells with access to open environment. Chain migration involves a more dynamic and less frequent interaction between cells. Chain migrating cells also restrict membrane process extension to the lead edge, maintaining contact with neighboring cells. B. Representative images of zebrafish neural crest cells exhibiting different migratory behaviors.

Figure 1.8. Contact Inhibition of Locomotion during Cell Migration. Contact inhibition of locomotion between single cells leads to collapse of cell protrusion and a change in the direction of migration.

27 Cranial Neural crest cell also encounter a physical barrier during their migration. The otic placode is positioned directly in front of the emigrating neural crest cells in mouse and chick and forces the cranial neural crest to alter their migration around the tissue and affects the shape of the sheet. However, this is not the case in zebrafish and frogs, as the otic placode is positioned such that the collective crest migration can occur directly adjacent to the tissue.

Migration of cranial neural crest is also negatively influenced by repulsive external cues that restrict the migratory path. These signals include Ephrines,

Semaphorins and Neuropilins, for which the neural crest cell has the corresponding receptors.

Trunk neural crest cells appear to migrate as streams of cells, converging from a broader region along the neural tube to form distinct segmental paths.

Trunk neural crest cells migrate from more caudal neuroepithelium along the dorsal neural tube. Trunk neural crest cells migrate along two distinct paths, either ventrally between the neural tube and developing somatic tissue or dorsolaterally in between the dorsal ectoderm and the dermomyotome. Trunk neural crest cells that enter the dorsolateral pathway give rise to pigment cells, whereas cells entering the ventromedial path give rise to the sympathetic and dorsal root ganglia, boundary cap cells, and Schwann cells (Theveneau and

Mayor, 2012).

However, unlike cranial neural crest cell migration, trunk neural crest cells display distinct differences in the timing and regulation of migration between species. In chick, the entry of crest cells into a migratory path is separated by a

24-hour period, with migration occurring medially prior to dorsolaterally. In

28 contrast, mouse trunk neural crest cells enter paths medial and dorsal simultaneously. In zebrafish, trunk neural crest cell migration along the medial path precedes dorsolateral migration by 4 hours (Raible and Eisen, 1996; Raible et al., 1992). Interestingly, entry along the dorsolateral path is restricted to cells of the melanoblastic lineage in chick and mouse, however in frog and zebrafish pigment cells migrate along both paths (Kelsh et al., 2009). It is unclear whether melanocyte entry along the medial path is due to less restrictive early fate determination or an alternative mechanism for regulation of dorsolateral path entry.

Trunk neural crest cell migration along the medial pathway is regulated by inhibitory environmental cues. In chick and mouse, inhibitory signaling through

Ephrin / Eph receptor dependent drive trunk neural crest cells migration along the rostral portion of the somite tissue (Baker and Antin, 2003; Bronner-Fraser et al., 1995; De Bellard et al., 2002; Krull et al., 1997). Further,

Semaphroin/Neuropillin (Schwarz et al., 2009) and Wnt-MuSK (Banerjee et al.,

2011) signaling also restrict trunk neural crest cell migration along the rostral portion of the somite. However, zebrafish trunk neural crest cells migrate through the medial portion of the somite and the restrictive influence of

Ephrin/Eph and Semaphorin/Neuropillin has not been assessed.

Migrating cells establish a front-rear polarity wherein membrane protrusion localize to the front end probe the environment and anchor forward movement while rear membrane adhesions are destabilized and membrane contracted (Petrie et al., 2009). Cells establish a front-rear polarity through distribution of the cytoskeletal proteins, including Rac1, RhoA and Cdc42 (Petrie

29 et al., 2009). Pard3 interacts with cytoskeletal regulators and has polarized localization during cell migration. During in vitro wound healing assays using

HeLa and Vero cells Pard3 was shown to localize along the leading membrane of individual migrating cells (Nakayama et al., 2008). In migrating fibroblasts,

Pard3 is localized at the leading edge of the cell and mediates protrusive activity through recruit Tiam1 to the leading edge (Mertens et al., 2006; Pegtel et al.,

2007; Wang et al., 2012). Tiam1 and Par complex interactions results in the accumulation of active Rac at the leading edge (Nelson, 2009; Nishimura et al.,

2005; Wang et al., 2012). In contrast, during in vitro wound healing assays using

MDCK and NIH3T3 cells Pard3 was shown to maintain the centrosome positioning and localized at cell-cell contact sites, lacking discernible enrichment at the leading edge of migrating cells (Schmoranzer et al., 2009). The role of

Pard3 during directed cell migration remains unclear. In Chapter II, the role of

Pard3 as a mediator of CIL during neural crest cell migration in vivo is discussed.

Schwann Cell Myelination

Schwann cells form the myelin sheath that surrounds peripheral axons.

The myelin sheath functions as a segmental insulator, restricting axon membrane depolarization to internodal segments termed nodes of Ranvier. Restriction of membrane depolarization results in fast, salutatory nerve impulse conduction along an axon. The myelin sheath is one of the most well characterized mammalian membranes due to the significant role myelin plays in human health and as a result of the abundance of myelin membrane and the ease of isolation.

Axonal sorting and myelination are complex cellular behaviors that involve

30 dynamic changes in Schwann cell morphology and gene expression. The mechanisms that regulate these changes in Schwann cell behavior are discussed in this section.

The transition from Schwann cell precursor to immature Schwann cell is characterized by the initiation of axon radial sorting. Whereas Schwann cell precursors loosely associate along nascent axons, immature Schwann cells ensheath axon bundles. Immature Schwann cells radially sort the ensheathed axons by extending their membrane processes into the bundle and associating along multiple small caliber axons or segregating out single large caliber axons

(Webster et al., 1973).

The process of radial sorting relies on Schwann cells forming a basal lamina to interpret instructive cues from the external environment and adjust their internal developmental programing accordingly. Immature Schwann cells that have defects in genes associated with establishing a basal lamina, such as focal adhesion kinase (FAK), cdc42, laminin-2, laminin-8 and nf2/merlin fail to complete the radial sorting process (Benninger et al., 2007; Chen and Strickland,

2003; Grove et al., 2007; Nodari et al., 2007; Wallquist et al., 2005; Yang et al.,

2005; Yu et al., 2005). Similarly, immature Schwann cells that have defects in cytoskeletal regulatory protein, including the typical Rho-GTPase rac1 do not extend processes into the axon bundles and fail to complete radial sorting.

Interestingly, radial sorting defects in Schwann cells lacking laminins were improved after forced expression of Cdc42 or Rac1 (Yu et al., 2009), further supporting the connection between basal lamina and cytoskeletal regulation in radial sorting.

31 As a result of radial sorting immature Schwann cells adapt one of two distinct relationships with peripheral axons: formation of a Remak bundle around multiple axons or wrapping and myelination of a single axon. An immature Schwann cell associated along several small caliber axons will form a

Remak bundle by surrounding several axons with membrane (Griffin and

Thompson, 2008). An immature Schwann cell associated along a large caliber axon, generally larger than 1 µM in diameter, will wrap and form compact myelin around the axon. However, whether Schwann cells form Remak bundles or a myelin sheath is not determined exclusively by the diameter of the associated axon. Recent evidence demonstrated the fate of an immature Schwann cell along an axon is influenced by the amount of Nrg-1 expressed on the axon surface

(Michailov, 2004; Taveggia et al., 2005).

Schwann cells spirally wrap their membrane around an axon forming a myelin sheath. Rac1, a known regulator of the actin cytoskeleton, is involved in axonal sorting and myelination (Benninger et al., 2007; Nodari et al.,

2007).Further support for cytoskeletal regulation during myelination come from

Neural Wiskott-Aldrich syndrome protein (N-Wasp) which is required for myelin formation (Jin et al., 2011; Novak et al., 2011). Activation of the Schwann cell myelination pathway is regulated by several mechanisms. Initially, immature

Schwann cells form Remak bundles or are directed toward the myelination stage in response to the amount of Nrg-1 expressed on the axon surface, with higher levels of Nrg-1 required for initiation of myelin formation and myelin thickness

(Michailov, 2004; Taveggia et al., 2005). Further, signaling through the G protein-coupled receptor Gpr-126 is required autonomously by Schwann cells for

32 formation of Remak bundles and myelination, although the identity of the ligand and whether it is expressed by axons is not known (Monk and Talbot, 2009;

Monk et al., 2011).

In addition to extracellular signaling cues, Schwann cell myelination is regulated by transcriptional activity (Jessen et al., 2008a). The transcription factor Sox10 (SRY-related HMG-box 10) is required throughout Schwann cell development (Britsch et al., 2001; Finzsch et al., 2010; Jessen and Mirsky, 2005).

Sox10 is a positive regulator of the transcription Oct6 (octomer-binding transcription factor-6), which is necessary for activation of the myelination program (Jaegle et al., 1996; Jaegle et al., 2003). Together, Sox10 and Oct6 form a feed-forward loop to activate the transcription factor

/Krox20 (early growth response-2) (Jagalur et al., 2011; Reiprich et al., 2010).

Egr2/Krox20 is essential for Schwann cell myelination. In Egr2/Krox20 knock- out mice, Schwann cells fail to wrap axons and arrest prior to myelination

(Topilko et al., 1994). The activity of Sox10, Oct6 and Egr2/Krox20 leads to the suppression of myelination inhibitors, such as c-Jun and Notch, and the expression of numerous myelin genes (Jessen et al., 2008b; Srinivasan et al.,

2012; Woodhoo et al., 2009).

Myelin membrane in both the central and peripheral nervous systems is composed of cholesterol, phospholipids, glycolipids, and myelin-specific proteins.

Myelin specific proteins are necessary for the myelin membrane layers to form tight apposition to one-another during wrapping. In Schwann cells, Myelin Basic

Protein (MBP), Myelin protein zero (MPZ, also called P0) and Myelin Proteolipid

Protein (PLP) are examples of myelin genes necessary to form tight apposition.

33 Both MPZ and PLP are membrane-spanning proteins and serve as spacers between the apposing membrane layers, whereas MBP in intrinsically unstructured and localizes intercellular along the major dense line. Interestingly,

MBP interacts with Actin, Tubulin, Ca2+- Calmodulin and Clathrin (Boggs,

2006), suggesting MBP may have additional functions beyond myelin membrane organization. Myelin protein expression and transcriptional activity during

Schwann cell myelination have been extensively studied, however the influence of cell polarity on Schwann cell myelination has only recently become a prominent research focus.

Schwann cells maintain contact with an axon and an extracellular matrix and therefore must be able to distinguish these two separate domains for the appropriate transport of cellular cargo. Accordingly, myelinating Schwann cells are polarized molecularly and morphologically, both longitudinally from node to node and radially from axon to basal lamina (Özçelik et al., 2010; Salzer, 2003).

Recent studies have demonstrated that Schwann cells localize polarity proteins along the radial axis, forming apical and basolateral domains similar to epithelial cell polarity (Chan et al., 2006; Lewallen et al., 2011; Özçelik et al., 2010; Poliak et al., 2002). This polarized organization of the Schwann cell can localize molecules necessary to convey signals from axons to Schwann cells that promote myelination (Pereira et al., 2012). Consistent with this, Pard3 is asymmetrically localized at the Schwann cell – axon junction following interaction and is required to localizes the BDNF receptor p75NTR (neurotrophin receptor), which can convey myelin-promoting signals, for myelination (Chan et al., 2006).

34 Disease

Peripheral neuropathies are a class of disorders in which humans sustain damage to the peripheral nervous system that disrupts communication between the peripheral and central nervous system and can result in significant debilitating effects on mobility and sensory function. Peripheral neuropathies can result from the progressive loss of myelin from peripheral axons, commonly referred to as demyelinating neuropathies. The majority of demyelinating neuropathies are inherited and often result from genetic abnormalities within

Schwann cells. The most common demyelinating neuropathy is Charcot-Marie-

Tooth disease (CMT). CMT has been linked to genetic mutation in several

Schwann cell genes, including Egr2 and myelin genes MPZ and PMP22 (Suter and Scherer, 2003). Currently, no therapies have been discovered for the treatment of CMT disease. Further, the underlying cause(s) of myelin related diseases, including Multiple Sclerosis and rare leukodystrophies are often complex and poorly understood. The inability to currently address these diseases underscores the importance of investigating the molecular and cellular mechanisms that govern myelination to aid in the development and application of translational therapies for neuropathic disease.

Aim and Structure

Aim

In summary, Schwann cells undergo dynamic transitions in cellular morphology, behaviors and gene expression during their development. Schwann cell behavioral transitions require the activation of transcriptional differentiation

35 programs and the complex reorganization of the cytoskeletal structure and cell polarity. In this chapter, the relevant aspects of Schwann cell development, directed cell migration and cell polarity were discussed, highlighting some of the important factors that mediate changes in Schwann cell behavior. In this thesis, the problem of how Schwann cells regulate their behavioral transitions during development will be addressed. More specifically, this thesis focuses on the role of cell polarity protein Partioning Defective - 3 (Pard3) throughout Schwann cell development in vivo. How Pard3 functions in specific Schwann cell behaviors are examined in further detail, including how Pard3 functions during collective cell migration and the temporal control of the myelination program.

Structure

The first Chapter of this thesis contains an overview of the topics discussed, highlighting the specific cellular behaviors that occur during Schwann cell development. Chapter II of this thesis describes the role of Pard3 in Schwann cell development and its content has been recently accepted for publication in a peer-reviewed journal. Chapter III addresses the aspects of Pard3 regulation of

Schwann cell development not discussed in Chapter II and describes newly generated tools for future research. Lastly, in Chapter IV the results of this thesis are discussed and their relevance to our current understanding of Schwann cell development is explored.

36 CHAPTER II 1

PARD3 REGULATES CONTACT BETWEEN NEURAL CREST

CELLS AND THE TIMING OF SCHWANN CELL

DIFFERENTIATION BUT IS NOT ESSENTIAL FOR NEURAL

CREST MIGRATION OR MYELINATION

Abstract

Schwann cells, which arise from the neural crest, are the myelinating glia of the peripheral nervous system. During development neural crest and their

Schwann cell derivatives engage in a sequence of events that comprise delamination from the neuroepithelium, directed migration, axon ensheathment and myelin membrane synthesis. At each step neural crest and Schwann cells are polarized, implying important roles for molecules that create cellular asymmetries. In this work we investigated the possibility that one polarity protein, Pard3, contributes to the polarized features of neural crest and Schwann cells that are associated with directed migration and myelination. We analyzed mutant zebrafish embryos deficient for maternal and zygotic pard3 function.

Time-lapse imaging revealed that neural crest delamination was normal but that migrating cells were disorganized with substantial amounts of overlapping membrane. Nevertheless, neural crest cells migrated to appropriate peripheral targets. Schwann cells wrapped motor axons and, although myelin gene expression was delayed, myelination proceeded to completion. Pard3 mediates

1 This chapter of the thesis is based on our manuscript “Pard3 regulates contact between neural crest cells and the timing of Schwann cell differentiation but is not essential for neural crest migration or myelination”, Blasky et al. 37 contact inhibition between neural crest cells and promotes timely myelin gene expression but is not essential for neural crest migration or myelination.

Introduction

Establishing a functional peripheral nervous system requires the coordinated development of axons and myelin-forming Schwann cells. During embryonic development Schwann cells undergo dynamic changes in both tissue location and cellular behavior to associate along and myelinate peripheral axons.

Schwann cell precursors arise from delaminating neural crest cells along the dorsal neural tube. During delamination neural crest cells alter their cellular morphology by losing their apical membrane adhesion and detaching from the dorsal midline, establishing a rounded morphology along the basal side of the neuroepithelium (Ahlstrom and Erickson, 2009; Clay and Halloran, 2013).

Following delamination Schwann cell precursors undergo extensive migration to associate along target axons in the peripheral tissue (Bronner-Fraser and

LeDouarin, 2012). Association along target axons signals Schwann cell precursors to stop their migration and promotes differentiation into an immature Schwann cell state (Birchmeier, 2009; Woodhoo and Sommer, 2008; Woodhoo et al.,

2009). Immature Schwann cells initiate the process of radial sorting, wherein

Schwann cells determine whether to wrap multiple small caliber axons as Remak bundles or transition into a mature myelinating Schwann cell, which forms a single compact myelin sheath on large caliber axons (Jessen and Mirsky, 2005).

The dynamic changes in cell behavior that occur during Schwann cell development have been extensively studied, but the events that govern the transitions between these behaviors remain poorly understood.

38 The transitions in neural crest and Schwann cell behavior leading to peripheral nerve myelination are associated with changes in cell polarity

(Etienne-Manneville, 2008; Özçelik et al., 2010). Consistent with this, proteins required for regulating cell polarity have been implicated in neural crest and

Schwann cell development. One example is Partitioning-Defective 3 (Pard3), a member of the evolutionary conserved partitioning-defective (PAR) multiprotein complex required for many polarized cellular processes (Özçelik et al., 2010;

Pegtel et al., 2007) reviewed in (Chen and Zhang, 2013; Goldstein and Macara,

2007; Nance and Zallen, 2011; Thompson, 2013). Prior to neural crest delamination, Pard3 was localized to cell-cell adhesion complexes within the apical domain of neuroepithelial cells (Clay and Halloran, 2013; Takekuni et al.,

2003). Subsequently, during neural crest cell migration, Pard3 was concentrated at the membrane leading edge where it mediated contact inhibition between neural crest cells, which might contribute to their directed migration (Moore et al., 2013). In cell culture, Pard3 localized to the Schwann cell-axon interface and

Pard3 knockdown inhibited myelination (Chan et al., 2006; Lewallen et al., 2011;

Özçelik et al., 2010). However, an extensive genetic analysis of Pard3 function in vivo has not been carried out. Consequently, the degree to which Pard3-mediated cell polarity contributes to the development of neural crest cells and their

Schwann cell descendants remains unclear.

We hypothesized that Pard3 provides polarity information for Schwann cells that facilitates their migration, axon interaction and differentiation as myelinating cells. To test this hypothesis we used a loss of gene function approach in combination with time-lapse imaging of transgenic zebrafish

39 reporters that mark the Schwann cell lineage. Here we provide evidence that

Pard3 mediates contact inhibition between neighboring Schwann cells, but that this function is not necessary for Schwann cell migration to peripheral targets.

Additionally, we show that loss of Pard3 function delays but does not prevent myelin gene expression and myelination. Our data are most compatible with the possibility that Pard3 coordinates other molecular mechanisms that drive

Schwann cell development to ensure timely myelination.

Results

Neural Crest Delamination From the Neuroepithelium Proceeds

Normally in the Absence of pard3 Function

To investigate the role of Pard3 in regulating Schwann cell behavioral transitions we utilized pard3fh305 mutant zebrafish, which have a chemically induced point mutation that changes a tyrosine at amino acid position 203 to a stop codon. This mutation is predicted to truncate the protein after the conserved oligomerization domain and before the PDZ domains (Fig. 2.1A), which bind cytoskeletal regulator proteins, adhesion complex proteins, and Protein Kinase C, iota (Prkcı) (Wei et al., 2004). Three cDNA variants of the zebrafish pard3 locus have been described and are predicted to encode distinct protein isoforms (Fig.

2.1A) (Geldmacher-Voss, 2003; Trotha et al., 2006; Wei et al., 2004). The premature stop codon introduced by the pard3fh305 allele truncates all three predicted isoforms. At 5 days post fertilization (dpf) homozygous pard3fh305 mutant larvae produced by matings of heterozygous parents (Zpard3fh305) had a subtle upward body curvature (Fig. 2.1B) and most mutant animals survived to

40 adulthood. Genotyping confirmed that larvae with curved bodies were homozygous for the pard3fh305 allele (Fig. 2.1C).

To investigate development in the absence of maternal contribution of pard3, we raised homozygous mutant animals and crossed them to produce embryos lacking both maternal and zygotic pard3 function (MZpard3fh305).

MZpard3fh305 larvae at 5 dpf had shortened bodies and more pronounced body curvature when compared with wild-type and Zpard3fh305 larvae (Fig.2.1B).

Furthermore, MZpard3fh305 larvae failed to develop full swim bladders, and only approximately 10% survived past 12 dpf. Embryos and larvae produced by MZpard3fh305 females and receiving one wild-type pard3 allele from either wild-type or heterozygous pard3fh305 males (pard3fh305/+) appeared phenotypically normal (Fig. 2.1B). To confirm that loss of pard3 function is responsible for the morphological defects of mutant larvae, we introduced the transgene Tg(hsp70I:pard3-GFP) (Hudish et al., 2013), which expresses Pard3 fused to GFP (Trotha et al., 2006) under control of heat-responsive regulatory elements (Shoji et al., 1998). Repeated induction of Pard3-GFP expression using elevated temperature during the first three days of development suppressed the body curvature defects and partially rescued swim bladder formation (Fig.

2.1D,E). Together these observations indicate that Pard3 is required for viability but that embryonic development can proceed with only maternally contributed

Pard3.

Figure. 2.1. Characterization of maternal and zygotic pard3 functions.

42 Figure. 2.1. Characterization of maternal and zygotic pard3 functions. A: Schematic representation of zebrafish Pard3 isoforms. Each isoform has a conserved oligomerzation domain (CR), three PDZ domains (PDZ1-3) and a Prkcı binding domain (PBD). The pard3fh305 lesion, changing a tyrosine to a stop codon at amino acid position 203 (Y203*) occurs after the oligomerization domain and before the PDZ binding domains (red line). B: Images of 5 dpf wild-type, Zpard3fh305, pard3fh305/+, and MZpard3fh305 larvae. MZpard3fh305 larvae fail to form full swim bladders (arrow) and have a more extreme body curvature than Zpard3fh305 mutants. C: Genotyping test for the pard3fh305 allele. Heterozygotes generate bands of 155 base pairs (wild-type allele) and 68 and 87 base pairs, which appear as one band on the gel (mutant allele). Homozygous mutants, selected on the basis of body curvature phenotype, produce only the 68 and 87 base pair fragments. D: Representative images of 5 dpf MZpard3fh305 larvae either without (“negative”, top row) or with (“positive”, bottom row) the Tg(hsp70I:pard3-GFP) transgene. Larvae in left column are control, non-heat shocked and those in right column were heat shocked. Pard3-GFP expression rescued the body curvature (arrowheads) and swim bladder (arrows) phenotypes. E: Graph showing quantification of heat shock rescue experiment. Larvae were produced by crossing MZpard3fh305 females to pard3fh305/+; Tg(hsp70I:pard3- GFP) males. Non-heat shocked control and heat shocked groups therefore consist of approximately 50% MZpard3fh305 and 50% pard3fh305/+ larvae. Larvae were scored at 5 dpf for severity of body deformation and swim bladder formation. Control, n=211; heat shock, n=182.

43 Schwann cells are specified from neural crest cells, which arise by delamination of neuroepithelial cells from dorsal neural tube. Delamination can occur via several processes including asymmetric division, force generation, and down-regulation of cellular adhesion complexes (Ahlstrom and Erickson, 2009;

Berndt et al., 2008; Clay and Halloran, 2010; Theveneau and Mayor, 2012).

Pard3 mediates the formation and maintenance of apical cellular adhesion complexes within mouse and chick neuroepithelial cells (Afonso and Henrique,

2006; Takekuni et al., 2003) and, in zebrafish, Pard3 localizes along the apical domain of pre-migratory neuroepithelial cells (Clay and Halloran, 2013).

Therefore, we hypothesized that Pard3 mediates the timing of trunk neural crest cell delamination. If so, absence of Pard3 might result in premature neural crest cell delamination and migration. To test this hypothesis, we introduced the transgene Tg(sox10:memRFP) (Kucenas et al., 2008), which marks neural crest cells with membrane tethered RFP, into MZpard3fh305 embryos and used time- lapse microscopy to analyze neural crest exit from the dorsal neural tube. As previously described (Raible and Eisen, 1996; Raible et al., 1992), beginning at 18 hours post fertilization (hpf) cells exited from the dorsal neural tube in control embryos (n=7 embryos) and migrated away from the dorsal midline in an anterior-to-posterior progression (Fig. 2.2A). The timing of neural crest cell delamination and initiation of migration was similar in MZpard3fh305 embryos

(n=9 embryos) to that of control embryos (Fig. 2.2B). We conclude that Pard3 function within neuroepithelial cells is not required to regulate neural crest cell delamination from the neural tube.

Figure. 2.2. Schwann cells exit the dorsal neural tube on schedule in pard3 mutants. Panels show representative frames from time-lapse movies of trunk neural crest cells between 18-24 hpf. Views are from dorsal with anterior to the left. Numbers denote time elapsed, in minutes, from start of imaging. Embryos are siblings and imaged sequentially in the same chamber. A: Initiation of neural crest migration in a control pard3fh305/+ embryo. Neural crest cells are marked by sox10:memRFP expression. Arrows mark migration on the medial pathway between neural tube and somites and asterisks mark migration on the lateral pathway, across the dorsolateral surface of somites. B: Migration of neural crest in a MZpard3fh305 mutant embryo. The onset and pattern of migration is similar to the control. Scale bar = 50 µM.

45 Pard3 Promotes Contact Mediated Inhibition But Does Not Drive

Neural Crest Cell Migration

Subsequent to delamination, neural crest cells migrate along discrete pathways to populate peripheral tissues. Cranial neural crest cells exit the neuroepithelium and migrate toward the eye and pharyngeal arches forming distinct streams (Clay and Halloran, 2010; Klymkowsky et al., 2010; Kulesa et al.,

2010). Similarly, trunk neural crest forms columns or streams of cells as they migrate into the periphery. Recent data produced using pard3 antisense morpholino oligonucleotides in zebrafish indicated that loss of Pard3 function inhibited both cranial and trunk neural crest migration (Moore et al., 2013).

However, cranial and trunk neural crest migration appeared normal in

MZpard3fh305 mutant embryos. In particular, cranial neural crest cells formed distinct streams as they migrated from the neuroepithelium at 30 hpf, similar to control (Fig. 2.3A,B) and formed normal jaw structures by 5 dpf (Fig. 2.3C,D).

Trunk neural crest cells also migrated to their motor axon targets in control and mutant embryos (Fig. 2.3E,F). Therefore, our genetic analysis indicates that

Pard3 function is not necessary to drive neural crest cell migration. Although neural crest cells arrived at their normal destinations in MZpard3fh305 mutant embryos, we noted differences in organization of the neural crest population. In control embryos many migrating neural crest cells appeared to be loosely associated, with few contacts between them (Fig. 2.3A,E). By contrast, in

MZpard3fh305 mutant embryos migrating neural crest cells appeared to be more densely packed or to have more area of overlap (Fig. 2.3B,F).

Figure. 2.3. pard3 is not essential for neural crest migration.

47 Figure 2.3. pard3 is not essential for neural crest migration. A,B: Representative images of cranial neural crest marked by Tg(sox10:memRFP) reporter expression in 30 hpf control pard3fh305/+ (A) and MZpard3fh305 mutant (B) embryos. View is from lateral with anterior to the left and dorsal up. Arrows indicate migrating groups of neural crest cells. In some regions, indicated by the outlined boxes, neural crest cells appear more tightly packed in mutant than in control. ov, otic vesicle. C,D: Representative images of jaw structures at 5 dpf in wild-type (C) and MZpard3fh305 mutant (B) larvae. The jaw in the mutant larva appears similar to that of wild type. E,F: Representative frames captured from time-lapse movies of trunk neural crest marked by Tg(sox10:memRFP) reporter expression. Motor neurons and axons are marked by Tg(mnx1:GFP) expression (green) in the final frames of the sequences. Images are from lateral with anterior to the left and dorsal up. Numbers indicate time, in minutes, elapsed from beginning of imaging. Images were captured from 18-24 hpf. In the control embryo (E), neural crest cells form streams (asterisks) with little overlap at points of contact (arrows). By contrast, neural crest cells appear less organized and overlap more in the MZpard3fh305 mutant embryo (F). Scale bar for E,F = 100 µM.

48 We therefore examined confocal image stacks to assess the amount of membrane contact between neighboring cells. For analysis of cranial neural crest we focused on cells located outside of branchial arch streams. This confirmed that in control embryos neighboring cranial neural crest cells had only a few, thin membrane contacts (Fig. 2.4A) but that neural crest cells in MZpard3fh305 mutant embryos had substantial amounts of overlapping membrane (Fig. 2.4B). In the trunk, neural crest cells in control embryos were elongated in the direction of migration with neighbor cell contacts limited to a single point, indicative of chain migration (Fig. 2.4C). By contrast, trunk neural crest cells of MZpard3fh305 mutant embryos were less elongated and had more area of neighbor cell contact

(Fig. 2.4D).

To further validate this observation we performed time-lapse imaging and examined cell-cell interactions by analyzing membrane overlap. To determine the percentage of membrane overlap we traced individual cell membranes over a period of 1 hour during neural crest cell exit from the dorsal neural tube and examined membrane overlap following contact. In control embryos neighboring neural crest cells collapsed their membrane projections following contact and restricted membrane overlap either by altering their migratory path or limiting process extension along a neighboring cell (Fig. 2.4E, n= 24 cells). By contrast, in

MZpard3fh305 embryos neural crest cells frequently extended membrane processes across neighboring cells and maintained their migratory direction over and along neighboring cells despite contact (Fig. 2.4F, n= 30 cells).

Figure. 2.4. Pard3 mediates contact inhibition between neural crest cells. A-F: Individual neural crest cell tracings represented as colored objects. A,B: Representative images of cranial neural crest cell migration in control (A) and mutant (B) embryos. In some regions, neural crest cells appear more tightly packed in mutant than in control. C,D: Representative images of trunk neural crest cells in control (C) and mutant (D) embryos; lateral view. Neural crest cells form chains elongated in the direction of migration in the control embryo (C). By contrast, neural crest cells appear less elongated and to be less well organized as chains in the mutant (D). E,F: Representative frames captured from time-lapse movies of trunk neural crest cell delamination in control (E) and mutant (F) embryos. Views are from dorsal with anterior to the left. Numbers denote time elapsed, in minutes, from start of imaging. Embryos are siblings and imaged sequentially in the same chamber. Neural crest cells in control embryos (n=30) retract cell membrane upon contact with neighboring cells. By contrast, neural crest cells in mutant embryos (n=24) fail to retract cell membrane and maintain cell overlap (F). Scale bar, 25uM.

50 We quantified these behaviors by measuring the amount of overlap between adjacent cells. This revealed that whereas in pard3fh305/+ control embryos approximately 4% of the area of adjacent neural crest cells was overlapping (n=43 cells), in MZpard3fh305 embryos about 10% of the area of neural crest cells overlapped (n=47 cells; p<0.0001).

Migrating populations of cells often engage in contact inhibition of locomotion (CIL), which is characterized by the collapse of membrane projections upon contact with another cell and alteration of migration (Mayor and Carmona-Fontaine, 2010). The overlapping nature of neural crest cells in

MZpard3fh305 mutant embryos is consistent with the loss of CIL. However, Pard3 function during CIL is unclear. To determine if Pard3 is localized to the site of cell-cell contact in migrating neural crest cells and therefore a candidate for regulation of CIL, we induced Pard3-GFP expression using the Tg(hsp70l:pard3-

GFP) transgene. Pard3-GFP was evident as small aggregates within cells (Fig.

2.5A), similar to previously reported overexpression results (Buckley et al., 2013;

Mizuno et al., 2003). At 20 hpf, Pard3-GFP was localized along contacting membranes of neighboring neural crest cells, consistent with the possibility that

Pard3 regulates CIL (Fig. 2.5A). To investigate this further, we performed time- lapse imaging. This revealed that as neighboring cells came in contact, Pard3-

GFP became localized to the membrane at the contact point. This was followed by retraction of membrane protrusions and dispersion of the Pard3-GFP that had been concentrated at the point of contact (Fig. 2.5B). Together with our observations that neighboring neural crest cells have more membrane contact in the absence of Pard3 function, these data support the idea that localization of

51 Pard3 to points of cell contact promotes CIL. However, in contrast to previous conclusions (Moore et al., 2013) our data do not support the idea that Pard3- mediated CIL is a principal driver of neural crest cell migration.

Figure. 2.5. Pard3 localizes at transient points of contact between neural crest cells. A: Image of neural crest cells marked by sox10:tagRFP (white) and Pard3-GFP (green) (24 hpf; lateral view of trunk). Pard3-GFP is concentrated at points of contact between cells (arrows). B: Images captured from time-lapse movies of neural crest cells (24 hpf; dorsal view of trunk). Numbers indicate time elapsed since beginning of image sequence. Image sequence shows one neural crest cell extending to contact another followed by process withdrawal. Pard3-GFP clusters at the point of contact and then disperses following withdrawal. Scale bars, A = 10uM B = 25uM

52 pard3 Mutant Schwann Cells Myelinate Motor Axons

Upon association with peripheral axons, neural crest-derived Schwann cells change shape to ensheath axons with myelin membrane. Ensheathing

Schwann cells have features of apical-basal polarity, with adaxonal and abaxonal membrane having characteristics of apical and basal membrane, respectively

(Özçelik et al., 2010). Previous work implicated roles for Pard3 in both axon wrapping and myelination by Schwann cells. In particular, our own analysis using pard3 antisense morpholino oligonucleotides suggested that loss of pard3 function interferes with Schwann cell wrapping and myelination (Tep et al.,

2012) and Pard3 siRNA knockdown in cultured Schwann cells blocked myelination (Chan et al., 2006). We therefore reinvestigated Schwann cell wrapping and myelination in vivo in the absence of both maternal and zygotic pard3 function. To do so we created Tg(sox10:memRFP);Tg(mnx1:GFP);

MZpard3fh305 larvae, in which Schwann cells express RFP and motor axons are marked by GFP expression. At 4 dpf Schwann cells were elongated and tightly wrapped around motor axons in both control and mutant larvae, with no apparent differences in morphology (Fig. 2.6A,B). Therefore, we now conclude that Pard3 is not necessary for axon wrapping.

Next, we investigated myelination by using in situ RNA hybridization to detect expression of mbp, which encodes a major myelin protein. At 4 dpf,

Schwann cells along the anterior trunk motor roots and posterior lateral line nerve (plln) expressed mbp (Fig. 2.6C,D). In contrast, Schwann cells in

MZpard3fh305 larvae lacked expression of mbp along the motor roots, although mbp expression was present along the plln and in the central nervous system

53 (Fig. 2.6G,H). To determine if mbp expression was only delayed, we next performed in situ RNA hybridization using 4.5 dpf larvae. In this case, mbp expression at motor nerves of MZpard3fh305 larvae was similar to that of control larvae (Fig. 2.6E,F,I,J). These data indicate that Pard3 is not necessary for myelin gene expression in vivo but that it may promote the timing of the myelination program.

Additionally, we used electron microscopy to assess myelin formation. At

8 dpf, myelin was evident, although poorly compacted, in both control and

MZpard3fh305 mutant larvae (Fig. 2.7A,B). To determine if myelin could become compacted in the absence of Pard3, we examined myelination in the small fraction of MZpard3fh305 mutant larvae that survived past larval stage (Fig.

2.7C,D). At 50 dpf, motor axons were surrounded by multiple layers of compacted myelin membrane in both wild-type and MZpard3fh305 mutant fish

(Fig. 2.7E,F). We conclude that Pard3 is not essential for myelination, although it promotes its timely initiation during development.

Discussion

Cells fated to become myelinating Schwann cells undergo a stepwise sequence of events during development including delamination from the neuroepithelium, directed migration into peripheral tissue, axon ensheathment and synthesis of myelin membrane. At each step these cells have a distinct polarity. Prior to delamination they have the apical-basal polarity characteristic of neuroepithelial cells, during migration they have distinct leading and trailing edges and during axon wrapping and myelination features of apical-basal polarity again become evident. These dynamic developmental steps imply dynamic

54 functions of proteins that contribute to cell polarity. However, few studies have investigated the role of any polarity proteins during the entire Schwann cell progression in vivo. In this work we tested a hypothesis that the Par complex protein Pard3 regulates polarity necessary for directed migration, axon ensheathment and myelination by Schwann cells.

Genes that encode Par proteins, including Pard3, were first identified using forward genetic screens for embryonically lethal mutations that disrupt early developmental patterning events in nematodes (Kemphues et al., 1988) The fly ortholog of pard3, known as bazooka, was similarly identified in a forward screen for lethal mutations that altered larval cuticle pattern (Nusslein-Volhard et al., 1984). Mouse embryos also die midway through development, at least in part due to abnormal cardiac development (Hirose et al., 2006). We were therefore surprised that homozygous pard3 mutant zebrafish did not have dramatic morphological defects as embryos or larvae and that they survived to adulthood. The morphological defects of embryos and larvae produced by homozygous mutant adults was more severe than that of those produced by heterozygous animals and these individuals rarely survived past larval stage, indicating that maternal contribution of pard3 function is sufficient to sustain nearly normal development. This is an additional sentence to fill the rest of this page because I am tired of working on this formatting. The discussion section continues below.

Figure. 2.6. Schwann cells wrap motor axons but delay mbp expression in the absence of pard3 function.

56 Figure. 2.6. Schwann cells wrap motor axons but delay mbp expression in the absence of pard3 function. A,B: Representative images of control pard3fh305/+ (A) and MZpard3fh305 mutant (B) embryos, focused on a single motor root. Schwann cells are marked by sox10:memRFP (red) and motor axons are marked by mnx1:GFP (green). Schwann cells ensheath motor axons similarly in both control and mutant. Boxes show orthogonal views of motor nerves at the level of the dashed line. Arrows indicate ensheathed motor axons. C-J: Representative images of mbp RNA expression in wild-type and MZpard3fh305 mutant larvae. Views are from lateral of the trunk with anterior to the left and dorsal up. At 4 dpf, mbp expression is evident at motor nerves (mn, asterisks) (C) and the posterior lateral line nerve (pLLn) (D) of a wild-type larva. By contrast, no motor nerve expression is evident at motor nerves of a MZpard3fh305 mutant larva (E) although pLLn expression appears normal (F). At 4.5 dpf, mbp expression is similar in wild-type and MZpard3fh305 mutant larvae at both motor nerves and the pLLn (G-J). Scale bar = 15 µM (A,B), 100 µM (C- J).

Figure. 2.7. pard3 is not essential for Schwann cell myelination. A-B: Transmission electron micrographs of coronal sections through the trunk region of 8 dpf wild-type (A) and MZpard3fh305 mutant (B) larvae. Motor axons are pseudocolored blue. Areas indicated by dashed boxes are shown at higher magnification in insets. Multiple layers of myelin membrane are evident in both wild type and mutant. C,D: Lateral views of 50 dpf wild-type (C) and MZpard3fh305 mutant (D) zebrafish. Dashed white line indicates region of coronal sections obtained for electron microscopy. MZpard3fh305 mutants are shortened and display severe body deformation, manifesting as a variable curved body axis. E-F: Transmission electron micrographs of coronal sections through the trunk region of 50 dpf fish. Areas indicated by dashed boxes are shown at higher magnification in insets. Myelin ultrastructure is similar in wild type (E, E’) and MZpard3fh305 mutant (F, F’). Scale bars, A,B,C,D,1 µM; A’,B’,C’,D’, 0.25 µM

58 Pard3 localizes to tight junctions at the apical/lateral boundary of vertebrate epithelia (Izumi et al., 1998) and promotes tight junction assembly

(Chen and Macara, 2005) Neural crest cells undergo an epithelial to mesenchymal transition (EMT) as they delaminate from the neuroepithelium, which is accompanied by disassembly of cellular adhesions such as tight junctions (Hay, 1995; Powell et al., 2013). Down-regulation of some tight junction components coincides with neural tube closure and neural crest delamination (Sauka-Spengler and Bronner-Fraser, 2008) and knock-down of the tight junction protein Cingulin enlarged the migratory neural crest population (Wu et al., 2011). We therefore considered the possibility that Pard3 similarly influences neural crest delamination. However, no abnormalities in the timing of neural crest delamination nor of the size of the neural crest population were evident in MZpard3fh305 mutant embryos, indicating that the timely onset of neural crest migration in zebrafish does not require modulation of Pard3 function.

As epithelial cells transform to migratory cells following EMT, apical-basal polarity changes to front-rear polarity (Nelson, 2009). Pard3 might contribute to front-rear polarity to facilitate directed migration, because knocking down Pard3 function impaired chemotaxis of isolated keratinocytes (Pegtel et al., 2007).

However, most neural crest cells do not migrate as individuals, but as cohorts of cells that engage in transient contacts with one another. Upon contact, neural crest cells withdraw protrusions and frequently change directions (Carmona-

Fontaine et al., 2008), a behavior known as contact inhibition of locomotion

(Abercrombie and Heaysman, 1953). CIL has been implicated as an important

59 driver of neural crest migration (Carmona-Fontaine et al., 2008; Matthews et al.,

2008; Theveneau and Mayor, 2010; Theveneau et al., 2013). Recent data provided evidence that Pard3 localizes to the point of contact between neural crest cells and that an approximately 50% reduction of Pard3 levels by antisense morpholino oligonucleotide injection in frog and zebrafish embryos blocked CIL and migration of cranial and trunk neural crest (Moore et al., 2013). Our data, drawn from analysis of embryos lacking both maternal and zygotic Pard3 functions, are in good agreement with the conclusion that Pard3 mediates CIL because neural crest cells had substantially increased amounts of overlapping membrane. However, our data do not support the conclusion that Pard3- mediated CIL is an important driver of neural crest migration. Although both cranial and trunk neural crest cells were abnormally organized in mutant embryos, they nevertheless migrated normally. In particular, formation of the jaw, which requires long-distance migration of many cranial neural crest cells, was patterned normally in Pard3 mutant embryos.

Neural crest-derived Schwann cells undergo one final reorganization of polarity upon completion of migration by transforming front-rear polarity back to apical-basal polarity (Özçelik et al., 2010). Consequently, the Schwann cell membrane in contact with the axon, known as the adaxonal membrane, has characteristics of apical membrane whereas the outer abaxonal membrane has features of basolateral membrane. This organization could have at least two important consequences for Schwann cell differentiation and myelination. First, juxtaposition of abaxonal-apical membrane to the axon could place molecules that facilitate axon ensheathment. Consistent with this, we previously concluded

60 that loss of Pard3 function interfered with the ability of Schwann cells to tightly wrap motor axons (Tep et al., 2012). However, we now think that effect was likely an artifact of morpholino oligonucleotide injection because in this study we found no evidence of abnormal ensheathment. Second, juxtaposition of abaxonal-apical membrane to the axon could localize molecules that convey signals from axons to

Schwann cells that promote myelination. Consistent with this possibility, Pard3 localized to abaxonal membrane and recruited p75 neurotrophin receptor, which can convey myelin-promoting signals (Chan et al., 2006). Knock down of Pard3 in cultured Schwann cells using short-hairpin RNA blocked expression of MBP, indicating that Schwann cell polarity is a critical feature of myelination (Chan et al., 2006). However, our in vivo analysis also fail to fully support this conclusion.

Although mbp transcription was delayed in larvae lacking maternal and zygotic

Pard3, the delay was slight and mature myelin appeared to be fully formed and compacted. Multiple signal transduction mechanisms promote Schwann cell myelination (Glenn and Talbot, 2013; Pereira et al., 2012; Salzer, 2012) and we speculate that these function normally even in the absence of Pard3.

In summary, our data support the idea that regulation of polarity contributes to migration and differentiation of neural crest and its derivative

Schwann cells. However, our data point to a more nuanced role for polarity mechanisms than suggested by data derived from in vitro models and use of knock down methods for loss of function studies. We propose that Pard3 plays a modulatory role by organizing some of the many signaling mechanisms that influence neural crest migration and Schwann cell differentiation.

61 Experimental Procedures

Zebrafish Husbandry

The animal work in this study was approved by the Institutional Animal

Care and Use Committee at the University of Colorado School of Medicine.

Embryos were produced by pair-wise mating and kept at 28.5°C in egg water or embryo medium. Embryos were staged to hours post fertilization (hpf) or days post fertilization (dpf) according to established zebrafish guidelines (Kimmel et al., 1995). Homozygous zygotic mutants for the pard3fh305 allele were created by pair-wise mating of pard3fh305/+ adults. Homozygous maternal zygotic mutants were created by pair-wise mating of pard3fh305/fh305 female and pard3fh305/+ male adults. The experiments conducted in this study used the following strains of zebrafish: Tg(sox10:memRFP)vu234 (Kucenas et al., 2008), Tg(hsp70l:pard3-

EGFP)co14 (Hudish et al., 2013), Tg(mnx:GFP)ml2 (Flanagan-Steet et al., 2005) and Tg(sox10:tagRFP)co26.

Generation of Tg(Sox10(7.2):tagRFP)co26

The Tg(Sox10(7.2):tagRFP) construct was generated using Gateway

(Invitrogen) recombination and inserted into a Tol2 Kit destination vector for microinjection (Kwan et al., 2007). Microinjected embryos were screened and transgene positive embryos were raised. Adults were mated with AB fish and progeny were screened for tagRFP expression and positive embryos were raised to establish transgenic lines.

62 pard3fh305 Genotyping

Embryos and larvae were genotyped using primers FWD: 5’-

ATTGGCTTCAGCAGTTTTAAGAAA-3’ and REV: 5’-

ATGATTGGCACTGAGTGAAGAAC-3’ and PCR to amplify a 155 base-pair (bp) product for. The PCR products were digested with HpyCH4IV (New England

Bioscience). The wild-type allele remains undigested and mutant allele is digested into 87 bp and 68 bp. The complete protocol is available at the Zebrafish

International Resource Center (http://zebrafish.org).

Time-lapse Imaging

At 18 hpf, embryos were embedded in low melting point agarose and mounted in a heated chamber (28.5°C) of a motorized stage. Z-stack images were obtained every 5-7 minutes from 18 to 25 hpf using a PerkinElmer UltraVIEW

VoX Confocal System coupled with a Zeiss Axio Observer inverted compound microscope fitted with a 20X objective. Using Volocity software (PerkinElmer,

Waltham, MA, USA) images were processed using deconvolution and contrast enhancement. Four-dimensional volumes were assembled and exported as

QuickTime movie files.

Tg(hsp70l:pard3-EGFP) Heat Shock Procedure

Either non-transgenic pard3fh305/+ or Tg(hsp70l:pard3-

EGFP);pard3fh305/+ males were mated to pard3fh305/fh305 females and the resulting embryos were spilt, with half untreated and half heat shocked for 30 minutes at

38°C in approximately 100 mililiters of egg water at 24, 48, and 72 hpf.

63 Transgene expression was confirmed by screening for GFP. For rescue analysis,

Tg(hsp70l:pard3-EGFP)+ larvae from non-heat shocked control and heat shocked groups were scored for the severity of the body deformation (Normal,

Mild, or Severe) at 4 and 5 dpf.

Cell Membrane Overlap Analysis

Individual cells within 40 micron Z-stack volumes were traced using

Volocity software v6.1.1. Total area for individual cells and cell overlap were determined using the Volocity. Statistical analysis of membrane overlap data was performed using Prism 6 (GraphPad) and two-tailed Student’s t-test.

Whole Mount Cartilage Staining

5 dpf larvae were anesthetized and fixed overnight in 4% paraformaldehyde solution (PFA). Cartilage was stained with Alcian blue as previously described

(Walker and Kimmel, 2007). Larvae were mounted in glycerol and bright-field images were taken using a Leica stereoscope.

In situ RNA Hybridization

Whole-mount in situ RNA hybridization was performed as described

(Thisse and Thisse, 2008) to detect mbp (Brösamle and Halpern, 2002).

Following hybridization, tissues were fixed with 4% paraformaldehyde, equilibrated in 70% glycerol and mounted on glass coverslips for whole-mount imaging. Images were collected using a Zeiss Axio Observer equipped with DIC optics, Retiga Exi digital color camera and Volocity software. All images were

64 imported into Adobe Photoshop software and image processing was limited to changes in levels, contrast, brightness and cropping.

Transmission Electron Microscopy

Juvenile fish at 8 and 50 dpf were anesthetized with tricaine, placed on ice, and fixed in a solution of 2% glutaraldehyde, 4% paraformaldehyde and 0.1 M sodium cacodylate, pH 7.4. Membranes were enhanced using either secondary fixation with OsO4, uranyl acetate, and imidazole, or secondary fixation using

OsO4-TCH-OsO4. Electron micrographs were collected using a FEI Techai G2

BioTwin microscope, transferred to Adobe Photoshop and image processing was limited to contrast and cropping.

65 CHAPTER III

PAR COMPLEX PROTEINS IN SCHWANN CELL DEVELOPMENT

Introduction

Pard3 is a highly conserved scaffolding protein that acts as a signaling hub, mediating cytoskeletal organization and cell polarity. Pard3 interacts with multiple proteins through binding along its PDZ domains, including fellow members of the Partioning Defective (Par) family of polarity proteins, Pard6 and

Prkcı. Together, Pard3, Pard6, and Prkcı form the Par polarity complex, which functions in the establishment and maintenance of cell polarity in a variety of specialized cellular behaviors. Loss of Pard3 function disrupts the timing of myelin gene expression during Schwann cell myelin formation (Discussed in

Chapter II). However, little is known about how Par complex members Pard6 and

Prkcı function during Schwann cell development, and how loss of Pard6 or Prkcı function would affect myelin formation.

Although the function of Par complex proteins in Schwann cell development is unclear, their function during the establishment of neuronal cell polarity has been previously described. Neurons form two morphologically and molecularly distinct domains, the axon and dendrite. The separation of the axon and dendrite domain creates a polarity along the neuron that is the basis for signal transduction across an axon. Par complex proteins are essential for the distinction between the axon and the dendrite domains through their polarized enrichment at the site of the future axon (Shi et al., 2003). Specifically, Pard3 localization facilities local activation of small GTPases and regulation of the actin cytoskeleton necessary for axonal growth and dendritic spine formation

66 (Schwamborn and Püschel, 2004; Schwamborn et al., 2007a; Schwamborn et al.,

2007b; Zhang and Macara, 2006). Further, Pard3 regulates microtubules organization and stability in axon formation (Chen et al., 2013). Previously, we demonstrated the loss of Pard3 function disrupted Schwann cell myelin formation along motor axons. Schwann cells rely on axonal signaling throughout their development, including initiation of myelination, suggesting the delayed myelin phenotype could result from neuronal defects.

Despite extensive study of the effects polarity protein expression and localization enact, the molecular mechanisms that regulate polarity protein expression and localization remain poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that mediate degradation or translational inhibition of target mRNAs and are required for Schwann cell development (Bartel, 2009;

Bremer et al., 2010; Yun et al., 2010). miRNAs have been shown to regulate the expression of polarity genes in vivo, including Pard3 and Prkcı (Hudish et al.,

2013). In zebrafish, miR-219 targets the 3’UTR region of both Pard3 and Prkcı mRNA. Knockdown of miR-219 results in prolonged maintenance of apical structures and deformation of the jaw (Hudish et al., 2013), unpublished observation). Interestingly, cranial neural crest cells migrate to and contribute in the formation of cranial structures, including the jaw, and disruption of Pard3 function was recently shown to inhibit cranial crest migration. Whether miR-219 knockdown phenotypes result from disrupted polarity protein function in migratory cranial crest has not been addressed.

The dynamic regulation of cell polarity during neural crest cell migration is a complex and unexplored area of research. Understanding how an array of

67 migratory cues are integrated both spatially and temporally to generate directional movement can provide great insight into normal morphogenetic events during development and pathological manifestations, such as cancer metastasis. Recent advances in molecular manipulations and microscopy have resolved many of the mechanisms of cell migration, yet the role of polarity proteins during migration remain poorly understood. Here, I introduce two novel transgenic zebrafish lines that provide powerful new tool for investigating the timing and molecular pathways involved in the dynamic regulation of cell polarity during migration.

Results

Schwann Cells Express mbp in Par Complex Mutants

Par polarity proteins can act individually or as a protein complex during development to establish and maintain cell polarity in a variety of specialized cellular behaviors. The Par polarity complex is composed of Pard3, Pard6 and

Prkcι (also known as aPKC). Disruption of Par complex member Pard3 during

Schwann cell development results in aberrant migratory behavior and delayed expression of myelin genes (Discussed in Chapter II). Given these results, the question of additional Par polarity proteins functions in Schwann cell myelination became relevant. Failure of Pard6 and Prkcı to function normally results in disruption of polarized cellular behaviors, including cell adhesion and migration (Goldstein and Macara, 2007).

To determine whether loss of Pard6 or Prkcı affected Schwann cell development in vivo, either through disruption of neural crest cell migration to

68 target axons or failure to myelinate, a loss of gene function approach was undertaken in combination with analysis of the expression of mature myelin gene myelin basic protein (mbp). Mutants lacking Pard6 or Prkcı function during

Schwann cell development expressed mbp in the central nervous system and along motor axons at 4 dpf, indicating that Schwann cells were able to delaminate, migrate and initiate myelination in these larvae (Fig. 3.1). However, mutants with disrupted Pard3 did not express mbp along motor axons and were to examined in further detail (discussed in Chapter II). These results provide insight into the requirement for specific polarity proteins during specialized cell behaviors, supporting previous evidence regarding the function of polarity proteins in Schwann cell development.

Motor Neuron Morphology Unchanged in Pard3 Mutants

One potential explanation for the delayed myelin gene expression observed in pard3fh305 mutant Schwann cells is disruption of motor neuron development. Motor neurons are specified in the ventral neural tube and extend axonal processes into the peripheral tissue. Neuronal specification involves asymmetric localization of polarity proteins, including Pard3 and therefore, may be disrupted in pard3fh305 mutants. To determine whether loss of Pard3 function in pard3fh305 mutant zebrafish affects neuron development, we analyzed axonal branching patterns using Tg(mnx1:GFP);MZ pard3fh305, which label neurons with GFP (Flanagan-Steet et al., 2005).

Figure 3.1. Schwann cells delay mbp expression in the absence of pard3 function. A-C: Representative images of Par complex member prkci (A), pard6 (B), and pard3 (C) mutant zebrafish. A’-C’: RNA in situ hybridization for myelin basic protein expression in Par complex mutant zebrafish trunk at 4 dpf. Schwann cells in prkci (A’) and pard6 (B’) mutant zebrafish express myelin basic protein along motor axons (arrows). Schwann cells in pard3 (C) mutant zebrafish do not express myelin basic protein at 4dpf.

70 In MZ pard3fh305 larvae, motor neuron axon branching patterns appeared similar to control larvae (Fig. 3.2), indicating motor neurons were specified and able to establish cell polarity for axon formation. Further, the number of motor neurons was quantified in transverse sections using Tg(mnx1:GFP);MZ pard3fh305. To distinguish motor neurons from sensory neurons, we counted the

GFP+ cells located within the ventral spinal cord during peripheral nerve development. At 3 and 5 dpf the number of MNX1+ cells in MZ pard3fh305 larvae was consistent with control siblings.

mir-219 and Jaw Formation in Pard3 Mutants

MicroRNAs (miRNAs) are essential for Schwann cell development and have recently been shown to regulate Pard3 and Prkcı expression in vivo (Bremer et al., 2010; Hudish et al., 2013; Yun et al., 2010). Loss of miR-219 in zebrafish produces a severe jaw deformation, potentially from elevated levels of Pard3.

Further, knockdown of Pard3 function in zebrafish was recently shown to disrupt cranial neural crest cell migration (Moore et al., 2013).

To determine whether the jaw phenotype produced by miR-219 loss results from elevated Pard3, I injected miR-219 MO into MZpard3fh305 embryos and examined jaw formation at 5 dpf. MZpard3fh305 miR-219 MO injected embryos displayed severe jaw deformation similar to wild-type injected embryos, in contrast to non-injected wild-type and MZpard3fh305 controls (Fig. 3.3).

Figure 3.2. Motor neuron specification and branching appear normal in the absence of pard3 function. Representative images of lateral view of zebrafish trunk at 4 dpf. Motor axon branching is visualized using Tg(mnx1:GFP) and appears similar in wild type (Top) and mutant larvae (Bottom) at 4 dpf.

Figure 3.3. Knockdown of miR-219 disrupts jaw formation. Representative images of jaw formation in non-injected control (Left) and miR- 219 MO injected larvae at 5 dpf (Right).

73 (PH)AKT-GFP Enriched at Migratory Cell Leading Edge

Neural crest cells are highly migratory and exhibit both directional cell migration and collective cell migration behaviors. To further investigate the role of Pard3 during neural crest cell migration and examine PIP3 as a potential signaling mechanism associated with changes in cell polarity I generated a transgenically encoded PIP3 biosensor for in vivo live imaging. I created a tissue specific reporter by constructing Tg(sox10:PH3-Akt-EGFP) transgenic lines expressing the PIP3 binding domain of Akt fused to EGFP driven by sox10 regulatory DNA (James et al., 1996). The reporter, hereafter referred to as the Akt biosensor, becomes relocalized from the cytoplasm to the membrane upon formation of PIP3 (Fig. 3.4). EGFP expression is concentrated at the leading edge of neural crest cells during migration and to sites of cell-cell contact (Fig. 3.4), constant with previously reported PIP3 localization. Further, expressing a cytosolic RFP expressed by a Tg(sox10:TagRFP) transgenic reporter (abbreviated as sox10:RFP) in the same cells allowed us to quantify EGFP localization by calculating a fluorescence intensity ratio. These results confirmed that EGFP was more highly concentrated along the leading edge within migrating neural crest cell. This transgenic reporter will provide a powerful new tool for investigating the timing and molecular pathway way activation involved in polarized signaling during neural crest cell migration.

Figure 3.4. PIP3 activation is enriched at the migratory leading edge and sites of cell-cell contact. A. Image of migratory neural crest cells labeled by Akt biosensor and cytosolic RFP in a living larva. Arrows indicate site of cell- cell contact and concentrated PIP3 activity. B. Schematic representation of Akt biosensor membrane localization in response to PIP3 binding.

75 Pard3-GFP is Enriched in Neuroepithelial Cell Apical Domains In

Vivo

Collectively migrating cells often display contact inhibition of locomotion

(CIL), which is characterized by the collapse of membrane projections upon contact with another cell and alteration of migration (Abercrombie and

Heaysman, 1953; Theveneau et al., 2010). We found neural crest cells in

MZpard3fh305 mutant embryos displayed increased membrane overlapping during migration, consistent with the loss of CIL. However, Pard3 function during CIL is unclear. To determine if Pard3 is localized to the site of cell-cell contact in migrating neural crest cells and therefore a candidate for regulation of

CIL, I generated an inducible Pard3 reporter by constructing Tg(hsp70l:pard3-

GFP) transgenic lines expressing Pard3 fused to EGFP (Trotha et al., 2006) driven by the regulatory DNA of heat shock inducible element HSP70I. Following heat shock induction, Pard3-GFP was evident in all tissues and concentrated along epithelial cell apical domain (Fig. 3.5), similar to previously reported overexpression results (Buckley et al., 2013; Mizuno et al., 2003). At 30 hpf,

Pard3-GFP was localized along contacting membranes of neighboring neural crest cells, consistent with the possibility that Pard3 regulates CIL (Fig. 3.5A’).

Expression of the Pard3-GFP reporter varied from tissue to tissue. For example, gut epithelial cells maintained GFP four days following reporter induction and cells of the central nervous system had little expression 24 hours post induction.

Figure 3.5. Pard3-GFP is localized along neuroepithelial cell apical domains in vivo. A: Transverse section through the zebrafish forebrain at 30 hpf. Pard3-GFP expression (green) is concentrated in the apical domain of neuroepithelial cells along the forebrain ventricle and within developing eye (arrows). A’: In migrating cranial neural crest cells (red), Pard3-GFP is localized to cell-cell contact sites and along the leading membrane B-B’: Pard3-GFP expression (green) is enriched in the apical domain of neuroepithelial cells during neurulation. C: Pard3-GFP expression along the apical domain of zebrafish gut epithelial cells.

77 Discussion

Cells fated to become myelinating Schwann cells undergo a stepwise sequence of events during development including delamination from the neuroepithelium, directed migration into peripheral tissue, axon ensheathment and synthesis of myelin membrane. Analysis of Par complex protein Pard3 function during migration and myelination suggests a prominent role, yet few studies have investigated the role of any polarity proteins during the entire

Schwann cell progression in vivo. We addressed this gap using loss of function genetic analysis in a zebrafish Pard3 mutant and found surprising results

(Discussed in Chapter II).

The role of Par complex proteins Pard6 and Prkcı in Schwann cell development is also poorly understood. Here, using Pard6 and Prkcı loss of function mutant zebrafish we examined myelin gene expression at 4 dpf. We found Schwann cells located along motor axons express myelin gene mbp. These results indicate that neither Pard6 nor Prkcı function is necessary in Schwann cells for mbp expression, and suggest neural crest cell delamination, migration, and initiation of myelination are uninhibited. Additional studies are needed to more clearly address Pard6 and Prkcı function during earlier cell behaviors, including delamination and migration.

Finding myelin gene expression in Par6 and Prkcı mutant zebrafish focused attention onto Pard3 and its function during Schwann cell development.

Schwann cells form an intimate relationship with neuron during their development and rely on neuronal signals for survival and differentiation.

Neurons are highly polarized cells and Par complex proteins are essential for

78 their development. To address whether Schwann cell phenotypes found in

MZpard3fh305 mutants result from motor neuron defects, we analyzed

Tg(mnx1:eGFP); MZpard3fh305 mutant larvae, which label motor neurons with cytosolic GFP. We found that although mutants lacked Pard3 function motor neuron specification and branching morphology appeared normal at 1 and 4 dpf.

Our results indicate motor neuron specification and pathfinding in zebrafish can occur normally despite loss of Pard3 function. These results fail to support previous reports that Pard3 function is necessary for neuron specification

(Schwamborn and Püschel, 2004; Schwamborn et al., 2007b; Schwamborn et al.,

2007a; Shi et al., 2003). One potential explanation for the disparity comes from the recent analysis of the Pard3 conserved amino-terminal domain (CR1). The

Pard3 CR1 domain binds and organizes microtubules during neuronal polarization and, following knockdown of endogenous Pard3, expression of the

CR1 domain was able to rescue neuronal specification (Chen et al., 2013). The pard3fh305 genetic lesion introduces a premature stop codon following the CR1 domain and may allow for expression of a functional truncated Pard3 isoform.

Future analysis of pard3fh305 protein structure is needed to address this possibility.

miR-219 was previously identified as an important mediator of glial cell differentiation and myelination, and directly targeted polarity proteins Pard3 and

Prkci (Zhao et al., 2010; (Hudish et al., 2013). Here, we demonstrate that knockdown of miR-219 results in the deformation of the jaw structure. One potential explanation for this phenotype is loss of miR-219 inhibits cranial neural crest cell migration due to disruption of polarity protein function, as previously

79 reporter (Moore et al., 2013). Our analysis of miR-219 knockdown in Pard3 mutant embryos does not support this hypothesis, however analysis of miR-219 knockdown in Prkci mutants has not been addressed.

Building off of previous work that demonstrated the utility of a Pard3 fusion protein for in vivo analysis of cell polarity during zebrafish development

(Buckley et al., 2013), I generated an inducible Pard3-GFP transgenic zebrafish line. The inducible nature of Tg(hsp70I:Pard3-GFP) permits analysis of Pard-3 at later time-points than previously available through micro-injection of pard3 transcript into 1-cell zebrafish embryos. Further, expression levels of Pard3-GFP can be modulated through the timing and frequency of heat shock induction, which allows researchers to examine the effects of over expression on specific molecular and cellular behavior.

Understanding how Par complex proteins function in the coordination and integration of cellular polarity during a wide array of cellular behaviors, including cellular migration and myelination will enhance our understanding and improve our ability to generate therapeutics targeting neurodegenerative disease and cancer.

80 CHAPTER IV

DISCUSSION AND FUTURE DIRECTIONS

Summary

This body of work contributes to the fields of developmental biology and neuroscience by enhancing our current understanding of polarity protein function in the complex molecular and behavioral events of

Schwann cell development. I have identified the polarity protein Pard3 as a mediator of migratory neural crest cell contact inhibition of locomotion

(CIL) and demonstrated its dynamic localization to sites of cell-cell contact in vivo. I have also found that Pard3 functions in the timing of

Schwann cell myelin gene expression and clarified Pard3 requirement for

Schwann cell myelination in vivo. My work using genetic loss of function analysis in vivo supports a more nuanced role for Pard3 during myelination than previously described in vitro. Finally, I established two novel transgenic zebrafish lines that will enhance our ability to investigate cell polarity dynamics in a variety of cell types in vivo. In this chapter, I discuss the role of Pard3 in neural crest cell migratory behavior and

Schwann cell myelination and how Pard3 may be regulating these behaviors. I examine current knowledge regarding Pard3 function and speculate about how these functions may provide a framework for my results and guide future studies.

81 Pard3 Regulates Neural Crest Cell Contact Inhibition

Neural crest cells are a highly migratory population of cells that delaminate from the dorsal neuroepithelial tissue and migrate extensively throughout the embryo. Advances in time-lapse microscopy and in our ability to perform in vivo manipulations have revealed neural crest cells often migrate as collective cohorts of cells. Our current understanding of neural crest cell migration establishes cell-cell contact as a mediator of their migratory cell behavior during collective cell migration (Friedl and Gilmour, 2009; Rørth, 2012).

Contact inhibition of locomotion (CIL) is a potential mechanism for cell contact mediated migratory behavior, and has previously been shown to regulate neural crest cell migration in vivo (Carmona-Fontaine et al., 2008). Recently, the polarity protein Pard3 was proposed as a molecular regulator of CIL in neural crest cell migration through its inhibition of Rac-GEF Trio (Moore et al., 2013). Here, I demonstrate in vivo Pard3 function within migratory neural crest cell is necessary for CIL. Additionally, through the use of a newly generated transgenic line I demonstrate for the first time the dynamic localization of the Pard3 during contact of migratory neural crest cells in vivo (Fig. 2.5 A,B). Despite loss of Pard3 function and CIL, both cranial and trunk neural crest cells were able to migrate to peripheral targets and form peripheral structures. These results provide a framework for understanding the mechanisms that regulate neural crest migration and raise important questions about the impact of migratory cues on neural crest cells. Furthermore, it remains unclear how impactful CIL is during neural crest cell migration, and how in the absence of CIL neural crest cells are able to migrate to target tissues.

82 Directional cues from the environment are one potential explanation for why neural crest cells migrate to peripheral targets despite the loss of Pard3 function and subsequent disruption of CIL. Neural crest cell migration throughout the developing embryo is guided by a combination of factors, and loss of any single factor may not be sufficient to inhibit migration, as previously suggested for CIL (Carmona-Fontaine et al., 2008; Moore et al., 2013). These directional cues include permissive and inhibitory signaling from the migratory environment and cell-cell contact during collective cell migration. CIL may be a context dependent mechanism for directing neural crest cell migration that can be compensated for by alternative signals. Consistent with this idea, neural crest cells were recently shown to alter migratory behavior following transplantation into different migratory environments in vivo, suggesting environmental cues can alter migratory behaviors (Wynn et al., 2013).

Finding Pard3 is localized to the sites of neural crest cell contact during migration and that it functions in the regulation of contact inhibition provides further insight into the role of polarity proteins during cell-cell contact and collective cell migration. CIL is thought to regulate migratory behavior in a variety of different cell types, including neural crest cells and metastatic cancer

(Mayor and Carmona-Fontaine, 2010). My findings, along with recent data (Wynn et al., 2013) suggest that cell migration is dependent on additional factors beyond

CIL. Together, these results reinforce the importance of understanding the interplay between various signaling cues that determine the behavior of collectively migrating cells, including cell-environment and cell-cell contact.

83 Pard3 Mutant Schwann Cells Delay Myelin Gene Expression

Schwann cells promote rapid, saltatory conduction of action potentials along the axon by insulating peripheral axons within a myelin-rich sheath. The myelin sheath is one of the most well characterized mammalian membranes due to the significant role that myelin plays in human health and disease. The myelin membrane is comprised of lipids, cholesterol, and myelin specific proteins.

Myelin specific proteins promote the tight association between membrane layers as they concentrically wrap around the axon and form the myelin sheath.

Schwann cells activate myelin gene expression during the process of myelination and therefore, myelin gene expression can be considered as a positive indicator of

Schwann cell maturation and myelin sheath formation. Specifically, expression of the myelin gene myelin basic protein (mbp) is considered a marker of mature myelin, due to its late expression compared with other myelin genes.

Myelinating Schwann cells are highly polarized morphologically and molecularly. To form the myelin sheath, Schwann cells wrap their membrane concentrically around an axon, which establishes two distinct cellular domains: the outer abaxonal domain, which interacts with the basal lamina, provides structure, and relays signaling from the extra cellular matrix; and the inner adaxonal domain, which maintains contact with the axon and is important for axon-glial signaling and trophic support. To fulfill these different functions of these two domains, Schwann cells asymmetrically localize domain specific molecules, such as adhesion proteins, signaling receptors, and myelin genes.

In this thesis, we demonstrate that pard3fh305mutant Schwann cells exhibit delayed expression of the mature myelin gene mbp suggesting disruption in the

84 timing of the myelination program. One potential explanation for this is the failure to properly regulate signaling receptors necessary for axon-glia signaling.

Axonal signaling mediates various aspects of Schwann cell development and disruption of the signaling leads to aberrant Schwann cell development (Sherman and Brophy, 2005). During myelination Pard3 localizes the BDNF receptor p75ntr along the adaxonal domain during myelin initiation and knockdown of Pard3 was shown to inhibit myelination (Chan et al., 2006). Consistent with this possibility,

Pard3 interaction with the PI3K phosphorylation product phosphatidylinositol

3,4,5-trisphosphate (PIP3) was shown to regulate endocytosis and recycling of cell surface receptors. Pard3 is localized to the plasma membrane in the presence of high levels of PIP3 and together, they channel signaling receptors into recycling, rather than the degradation pathway (Laketa et al., 2014). Loss of

Pard3 may disrupt the endocytosis and recycling of Schwann cell signaling receptors, leading to their degradation. Further, degradation of signaling receptors during myelin initiation would require the synthesis and localization of new receptors to the axon glia surface, causing a delay in myelin gene activation.

This mechanism of PIP3-Pard3 mediated receptor recycling benefits the cell by limiting the degradation of physiologically relevant receptors, which decreases the requirement of transporting newly formed proteins to distant domains and occurs in similar polarized events, such as axonal growth and pathfinding and leading edge maintenance in migrating cells (Haugh et al., 2000; Shi et al.,

2003).

A second potential explanation for the delay in myelin gene expression is disruption of myelin membrane compaction during myelination dampening the

85 necessary signaling for myelin gene activation. Cytoskeletal components relays information that promotes changes in cell behavior and gene expression.

Disruption of cytoskeletal regulation may alter the ability of Schwann cells to activate mature myelin genes. Consistent with this mechanism, Pard3 interacts with cytoskeletal regulatory genes, including Rac1 and Ccd42, and has previously been shown to influence myelin sheath compaction in vivo (Tep et al., 2012).

Further, disruption of Rac1 or Cdc42 inhibits Schwann cell process extension and wrapping during radial sorting of axon. This mechanism would be consistent with the previous report of loose myelin sheath formation using pard3 MO knock down. Again, the caveat of MO experimentation is their eventual depletion and return of normal gene function. This would occur at the similar time to the initiation of myelination. It would be challenging to tease out the subtle delay in compaction of the myelin sheath in our model, as the limitations of confocal microscopy and the inability to descent distinct temporal changes in the myelin wraps through TEM analysis of the myelin ultrastructure. Further, Schwann cell specific loss of N-cadherin in vivo, which localizes Pard3 during Schwann cell myelination (Chan et al., 2006), results in a delayed myelination phenotype, consistent with my results (Lewallen et al., 2011).

Regulation of myelin formation remains a topic of great interest. Research into human diseases that result from demylination, such as CMT or multiple sclerosis, would benefit tremendously from understanding the mechanisms that regulate initiation of cell wrapping behaviors and myelin gene expression. In this thesis, I investigated the role of polarity protein Pard3 in Schwann cell myelin formation. The results discussed in the preceding chapters will help to inform the

86 myelin field through the demonstration that disruption of Pard3 is not sufficient to inhibit Schwann cell transition to mature myelin in vivo, as had been previously described in vitro. Our results suggest that Pard3 functions during myelin initiation and wrapping, but may be compensated for by an unknown alternative mechanism.

Future Directions

Analysis of pard3fh305 Mutant Allele

The results presented in this thesis provide several possible directions for future research and novel molecular tools to begin. Although we have linked the phenotypes found in MZpard3fh305 fish to the genetic point mutation which introduces a premature stop codon in exon 5 of the zebrafish par3 allele, the properties of the mutated Pard3 protein remain unclear. Lacking an antibody to unambiguously mark zebrafish Pard3 in situ and by Western blot analysis, we are unable to quantify changes in Pard3 expression or characterize structural Pard3 variants.

Zebrafish are an excellent model for investigation of gene function during development due to their rapid external development and their accessibility for observation and genetic manipulation. The use of morpholino anti-sense oligonucleotides (MO) is a prominent technique for analysis of gene function in zebrafish. MO are designed to either inhibit translation of a targeted transcript or to interfere with transcript splicing to produce a non-functional protein.

A Pard3 translation blocking MO is available and has been utilized in work from in our laboratory and others (Alexandre et al., 2010; Hudish et al., 2013;

87 Tep et al., 2012) to knockdown expression during early developmental events (0-

4 dpf). However, MO are exhausted after only a few days which limits analysis of later developmental events, including Schwann cell myelination. Further, the variability of MO injections and potential of off-target effects, which too often associated with knock down of the target gene function, bias perception of MO generated results.

Currently, two techniques are gaining momentum as alternatives to MO in zebrafish: Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs).

Both techniques involve binding and cleaving DNA in a sequence specific manner, which promotes gene mutations. Future analysis of Pard3 function in zebrafish may require utilizing these technologies to generate mutant alleles in a sequence specific manner, and refine our understanding of Pard3 structural domains.

Par Complex Proteins And Dynamic Migratory Cell Behavior

Neural crest cells migrate extensively during development and are an excellent model for investigating cell migratory behaviors in vivo. In this study I demonstrate Pard3 function within migratory neural crest cell is necessary for

CIL and further demonstrate the dynamic localization of Pard3 in vivo during cell-cell contact. These results raise intriguing questions about the nature of

Pard3 localization and its requirement for mediating behavior following cell-cell contact. Further, theses results highlight our limited understanding of Par polarity protein in the dynamic reorganization of cytoskeletal components during cell migration. Is Pard3 functioning as a signaling hub, binding and localizing

88 multiple effector proteins to the site of dynamic cell rearrangement, such as fellow Par complex proteins Pard6 and Prkcı? Future studies using currently available Par6 and Prkcı mutants in combination with in vivo time-lapse microscopy may reveal a yet uncharacterized Par complex dependent mechanism for cell migration. Further, significant advances have been made in establishing and quantifying fluorescently-labeled biosensors in vivo, including Rho, Rac1, and cdc42 (Kraynov et al., 2000; Nalbant et al., 2004; Pertz et al., 2006; Reffay et al., 2014). These tools in combination with the ease of genetic manipulation in zebrafish mutants provide an exciting model in which to study dynamic migratory behaviors.

Conclusion

The work presented in this thesis demonstrates the significance of Pard3 function in Schwann cell development. Our findings emphasizes the importance of understanding how localized polarity protein activity regulates such a wide range of cellular behaviors and highlight Pard3 as a potential hub for coordinating and integrating various signaling pathways in cell migration and myelination. These results open up a vast field for further study, as we attempt to better understand myelination, cell polarity, and Schwann cell development.

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