The Expression and Regulation of the Po Gene During Development of the Rat: Regulation of Basal Level and Early Expression

by

Meng-Jen Lee

Department of Anatomy and Developmental Biology

University College London

A thesis presented for the degree of Doctor of Philosophy

The University of London, 1998 ProQuest Number: U642130

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Acknowledgements

I wish to thank Professors Kristjan lessen and Rhona Mirsky for their guidance and enthusiasm throught the course of this work. I also thank Dr. Myrna Dent and Dr. Eric Parmantier for their help and numerous discussions, especially in in situ hybridization technique. I am grateful to Dr. Angela Brennan who contributed to the set up of the neural crest cultures and the study of the regulation of Po protein, and Dr. Ester Calle for contributing to the study of the lineage segregation. I thank Dr Louise Morgan, Dr Helen Stewart and Dr. Ziping Dong for their discussion and help, especially in the early days. I also acknowledge Charlotte Dean, Christiane Zoidl and Mary Rahman for technical support and making working in the lab easy.

I am grateful to Dr. V. Pachnis, Dr. D. Ish-Horowilz, Dr. C. Birchmeier, Dr. C. Dickson, Dr. Wilkinson, Dr. G. Lemke, Dr. I. Griffth, Dr. J. Lewis, Dr. D. Henrique for their gift of plasmids, Dr. B. Zalc for the PLP transgenic mice. I want to thank Dr. Jon Clark and Dr. Paul Martin for the micrscope and photography setup. Dr. Sue Manning for sharing her methods, Steve Harsum and C P Ma for their advice on computer methods.

Finally I thank my parents and my family for their support over many years. TABLE OF CONTENTS TITLE 1 ACKNOWLEDGEMENTS 2 TABLE OF CONTENTS 3 ABSTRACT 5 LIST OF TABLES AND ILLUSTRATIVE MATERIAL 7 ABBREVIATIONS 9 CHAPTER 1 : GENERAL INTRODUCTION 11 1.0 Table of contents 12 1.1 PNS glia 15 1.2 The molecular phenotypes of Schwann cells 18 1.3 Neuronal influence on Schwann cells 31 1.4 Wallerian degeneration 34 1.5 Myelination and the structure of myelinated fibres 36 1.6 Other Schwann cell functions 37 1.7 The Po molecule 37 1.8 The neural crest 42 1.9 Gliogenesis and neurogenesis during the formation of the dorsal root ganglion 53 1.10 Axon/Schwann cell interactions during the formation of nerves 55 1.11 The Schwann cell precursor 56 1.12 The CNS/PNS transitional zone 58 1.13 The gut 59 1.14 The placode 61 1.15 The ear 65 1.16 The nose 75 1.17 Illustrative material 81 CHAPTER 2: Materials and Methods 87 CHAPTER 3: The differential regulation of Po gene in the 2 mature phenotypes of the Schwann cell lineage 110 3.0 Table of contents 111 3.1 Introduction 112 3.2 Results 3.3 Discussion 119 3.4 Ilustrative material 121 CHAPTER 4: The study of Po gene expression in the migrating neural crest and its derivatives 126 4.0 Table of contents 127 4.1 Introduction 129 4.2 Results 135 4.3 Discussion 145 4.4 Illustrative material 158 CHAPTER 5: The study of Po gene expression in the development of the inner ear 168 5.0 Table of contents 169 5.1 Introduction 171 5.2 Results 175 5.3 Discussion 180 5.4 Ilustrative material 196 CHAPTER 6: General Discussion 201 CHAPTER 7 : References 208 Abstract This thesis represents a study of the Po gene, the major myelin protein gene in Schwann cells, during development of the rat. The major function of Po is in compaction of the myelin membrane during myelination, but I have found, using sensitive in situ hybridisation methods, that the gene is expressed much earlier than the onset of myelination. My experiments show that the Po gene is constitutively expressed early in development of the Schwann cell lineage, and that in adult nerves the Po gene is up-regulated and down-regulated differently in the two mature Schwann cell phenotypes. I found that Po mRNA is expressed at similar levels in Schwann cells at embryonic day 18, both in the sciatic nerve, where many Schwann cells will begin to myelinate shortly after birth, and also in the sympathetic trunk, where most Schwann cells are non-myelinating. Exploration of Po expression at earlier developmental stages, revealed that Po mRNA is expressed in Schwann cell precursors, the immediate precursors of Schwann cells in peripheral nerves, and also in a subpopulation of neural crest cells. I used the neural crest/ peripheral nervous system lineage, a relative simple model, to understand how cells of the neural lineage differentiated after they were generated from ectodermal cells in the process of neurulation. In the second part of my study, by correlating Po mRNA expression patterns in whole mount preparations and sections at embryonic ages from day 10 to day 14 with known neural crest migration patterns and with expression of neuronal and other neural crest lineage markers, I conclude that Po expression is restricted to a subpopulation of migrating crest cells which are likely to represent a cell population showing early commitment to the glial lineage. Special attention is paid to expression patterns in the forelimb and hind limb regions at embryo day 12, to expression patterns in the head at embryonic days 10 and 11, and to expression patterns in the developing gut at embryonic days 12-14. The onset of Po mRNA expression is compared with that of other molecules known to be specific to the Schwann cell lineage, and it is concluded that Po is the earliest well characterised marker of a glial phenotype. In the third part of the study I describe the unexpected expression of Po mRNA in the inner ear, where it has not previously been described. The expression of Po in the otic placode/pit stage at embryonic day 10, in the otic vesicle at embryonic day 12 and in the embryonic inner ear at later stages of development is described, and compared to several known molecular markers of the development of inner ear. The pattern of Po expression is broad but specific, with boundaries that clearly delineate segments of inner ear. Olfactory glial cells, which are placode derived, are Po positive during development. Po expression in olfactory glial cells persist through adulthood, although the olfactory nerves are not myelinated. List of Tables Table 1-1 Neural crest derivatives 43 Table 1-2 Derivatives of neural crest and placodes, categorised by cell type 63 Table 1-3 Contribution to the cranial ganglia by placodes and crest 63 Table 1-4 Combinatory code of transcription factors in placodes 65 Table 2-1 Restriction sites and polymerase used to generate riboprobes 109 Table 5-1 Comparison of neural crest and neurogenic placode 183 Table 5-2 The earliest innervation of inner ear 190 List of Illustrative Materials Figure 1-1 Molecular structure of Po protein 81 Figure 1-2 Diagrammatic representation of Po orientation in the myelin membrane 82 Figure 1-3 Neural crest migrating pathway in the trunk 83 Figure 1-4 Gross structure of inner ear 84 Figure 1-5 Structure of cochlea and Organ of Corti 85 Figure 1-6 recycling cells 86 Figure2-1 cDNA encoding the entire Po coding sequence 108 Figure3-1 Po mRNA in the neural crest and in developing sciatic nerves 121 Figure3-2 Axon-dependent and reversible down-regulation of Po mRNA in adult non-myelin-forming Schwann cells 123 Figure3-3 Removal of Schwann cells from axons leads to a fall in myelin related Po expression, while basal levels are relatively unaffected 124 Figure3-4 Schematic representation of Po mRNA distribution in cells of the Schwann cell lineage 125 Figure 4-1 RT-PCR of Po in developing embryo 158 Figure 4-2 Po expression in ElO 160 Figure 4-3 Po expression in El 1 ; comparison to TUJ-1 160 Figure 4-4 Po expression in E l2; comparison to TUJ-1, ErbB3 and Krox-20 160 Figure 4-5 Po expression in E l4 161 Figure 4-6 Developing enteric nervous system 162 Figure 4-7 Neural crest culture 164 Figure 4-8 The periodic pattern of Po expression in the migrating neural crest 165 Figure 4-9 A summary of Po expression during the generation of peripheral glial phenotypes 167 Figure 5-1 Po expression in El 1; comparison to Delta-1 194 Figures-2 Po expression in otic vesicle; comparison to PLP/DM-20 194 Figure 5-3 Po expression in E14 inner ear, serial sections 195 Figure 5-4 Po expression in utricle/cochlea/ganglion at E l4 196 Figure 5-5 Po compared to c-ret and BMP-4 in E l4 inner ear 197

Figure 5-6 Po compared to BMP-4 and serrate-1 in E l4 cochlea 199 Figure 5-7 Po compared to c-ret in E l4 utricle and cochlear 199 Figure 5-8 Po expression in developing olfactory nerves 200 ABBREVIATIONS 3’ UTR 3’ untranslated region ADS antibody diluting solution BDH dopamine-p-hydroxylase BMP-4 bone morphogenetic protein cAMP cyclic AMP CNPase 2’,3’,-cyclic nucleotide 3’-phosphodiesterase CNS central nervous system CNTF ciliary neurotrophic factor CSX cervical sympathetic trunk DMEM Dulbecco’s modified Eagle medium DREZ dorsal root entry zone DRG dorsal root ganglia

E() embryonic day ENS enteric nervous system Gal-C galactocerebroside GAP-43 growth associated protein 43 GDNF glial cell line derived neurotrophic factor GFAP glial fibrillary acidic protein IHC inner hair cells ISH in situ hybridization LIF leukaemia inhibitory factor MAG myelin-associated glycoprotein MBP myelin basic protein MEM minimum essential medium NCAM neural cell adhesion molecule NGF nerve growth factor NT-3 neurotrophin 3 NT-4 neurotrophin 4 OBECs olfactory bulb ensheathing cells OHC outer hair cells

P() postnatal day Po protein zero P2 myelin protein 2 p75NGF-R p75 the low affinity NGF receptor PBS phosphate buffered saline PF paraformaldehyde PLL poly-L-lysine PLP proteolipid protein PMP-22 peripheral myelin protein 22 PNS peripheral nervous system PSANCAM polysialyated NCAM RT-PCR reverse transcriptase polymerase chain reaction SCIP suppressed cAMP inducible POU protein SMP Schwann cell myelinating protein TV tegmentum vasculosum TZ transitional zone

10 Chapter 1 General Introduction

11 1.0 Table of contents 12 1.1 PNS glia 15 1.2 The molecular phenotypes of Schwann cells 18 1.2.1 Appearance and regulation of Schwann cell phenotypes during development 18 1.2.2 Non-myelinating phenotypes 19 1.2.3 Myelinating phenotypes 20 1.2.4 Molecules expressed by both phenotypes 25 1.2.5 Molecules not specific to Schwann cells but important in PNS myelination 26 1.2.6 Ion channels 29 1.3 Neuronal influence on the Schwann cells 31 1.3.1 Regulation of the lineage choice and embryonic development of the Schwann cell lineage; signalling by p-neuregulin binding to ErbB3 receptor 31 1.3.2 Axonal regulation of the Schwann cell phenotypes 33 1.4 Wallerian degeneration 34 1.5 The myelination and the structure of myelinated fibres 36 1.6 Other Schwann cell functions 37 1.7 The Po molecule 37 1.8 The neural crest 42 Table 1-1 Neural crest derivatives 43 1.8.1 The experimental approaches 44 1.8.2 The migrating pathways 45 1.8.3 Molecular markers for neural crest 48 1.8.4 Control of the migration pathway 49 1.8.5 The plasticity and the heterogeneity of the neural crest population 50 1.8.6 The lineage relationship of the neural crest derivatives 51 1.9 Gliogenesis and neurogenesis during the formation of dorsal root ganglion 53 1.10 Axon/Schwann cell interaction during the formation of nerves 55 1.11 The Schwann cell precursor 56 1.11.1 Schwann cell precursor is a major regulator of nerve development

12 during early development of PNS 57 1.12 The CNS/PNS transitional zone 58 1.13 The gut 59 1.14 The placode 62 1.14.1 Origin 62 1.14.2 Organogenesis 62 1.14.3 Contribution to cranial ganglion 62 Table 1-2 Derivatives of neural crest and placodes, categorised by cell type 63 Table 1-3 Contribution to the cranial ganglia by placodes and crest 64 1.14.4 Comparison of the placode and the neural crest 64 1.14.5 Molecular markers to date 64 Table 1-4 Combinatory code of transcription factors in placodes 65 1.15 The ear 1.15.1 General anatomy and corresponding functions of the inner ear 65 1.15.2 The structure of sensory organs and their cell types 66 1.15.3 The generation of the inner ear 67 1.15.3.1 Early morphological changes 68 1.15.3.2 Regional specification 68 1.15.3.3 The specification of sensory organ versus specification of other structures 70 1.15.4 Cell lineages of the inner ear 71 1.15.4.1 The neurogenic lineage 71 1.15.4.2 The sensory cells: hair cells and support cells 72 1.15.4.3 The lineage of melanocytes 72 1.15.4.4 Non-sensory, non-melanocyte epithelial cells 73 1.15.5 Factors responsible for the induction and differentiation of the otic vesicle 74 1.16 The nose 1.16.1 Terminology 74 1.16.2 An overview of the olfactory system 75 1.16.3 Plasticity in the olfactory system 1.16.3.1 Neurogenesis in the olfactory epithelium 76

13 1.16.3.2 The ability of olfactory axons to regenerate in the CNS 77 1.16.4 The origin and development of olfactory Schwann cells 77 1.16.5 The phenotypes of olfactory Schwann cells; a mixture of PNS and CNS phenotypes 79 1.16.6 The lineage of olfactory ensheathing cells: a mixture of two cell lineages or two phenotypes of a same lineage? 80 1.17 Illustrative material Figure 1-1 Molecular structure of Po protein 81 Figure 1-2 Diagrammatic representation of Po orientation in the myelin membrane 82 Figurel-1 Neural crest migrating pathway in the trunk 83 Figure 1-2 Gross structure of inner ear 84 Figure 1-3 Structure of cochlea and Organ of Corti 85 Figure 1-4 recycling cells 86

14 Po is the major myelin protein in the peripheral nervous system (PNS) and plays an important role in myelination. It has been used as a model to study myelination and axon-to-glia effects. It was always believed that Po is expressed postnatally only in myelinating Schwann cells, and the same was assumed in several transgenic experiments designed to use the Po promoter for Schwann cell specific, postnatal expression. A trend of discovering myelin genes during mid gestation stage (Ikenaka et al., 1992; Nakajima et al., 1993; Timsit et al., 1992; Yu et al., 1994; Zhang et al., 1995) points to a need to study Po expression at a stage when the parent cells of Schwann cells are generated from the neural crest. In this chapter the Po molecule itself, the regulation of molecules expressed in Schwann cells, and the development and lineage regulation of the cell types that are relevant are described.

1.1 PNS glia Chick-quail explant experiments (see 1.8 Neural Crest section) showed that neural crest cells proliferate and differentiate to form seemly unrelated types of cells, which include the glial cells of the sensory, sympathetic, and parasympathetic nervous system (Le Douarin and Smith, 1988; Weston, 1991). Schwann cells are derived from the neural crest via intermediate cells called Schwann cell precursors (lessen et al., 1994; see following). Other major PNS glial cells which are also derived from the neural crest include the teloglia associated with the somatic motor terminals, satellite cells associated with neuronal cell bodies in the ganglion, and the enteric glial cells in the enteric plexus of the gut (Georgiou et al., 1994; Gershon et al., 1993; Pannese, 1981). Not all PNS glial cells are derived from the neural crest. Some Schwann cells and satellite cells of the dorsal root ganglion (DRG) are derived from the neural tube (Carpenter and Hollyday, 1992; Lunn et al., 1987; S harm a et al., 1995). Olfactory bulb ensheathing cells (OBECs) located in the CNS/PNS transition zone and along the olfactory nerves are believed to be derived from the placode-derived olfactory epithelium (Chuah and Au, 1991, Doucette, 1989). For a general introduction of phenotypes of peripheral glia, the satellite cells and teloglia are described in the next section. Olfactory glia and enteric glia are detailed in Gut and Nose sections in this chapter respectively.

15 Satellite cells The perikaryon of a neuron in the DRG or autonomic ganglia is ensheathed by small, flat epithelium-like satellite cells that are continuous with Schwann cells enveloping the axon. Besides providing a continuous cover for neuronal bodies and their processes (Pannese, 1960; 1968), the satellite cells also secrete constituents of basal lamina to separate the glial cells and ensheathed neurons from other elements of the extracelluar matrix that are present in the ganglia (Pannese, 1968, Baluk et al., 1985). The position of the satellite cells relative to the neuronal cell bodies they ensheath changes slowly, possibly associated with synaptic remodelling (Pomeroy and Purves, 1988). The satellite cells share many similarities with Schwann cells. Like Schwann cells, they respond to axotomy by proliferation, but the response of the satellite cells is generally more rapid (Lu and Richardson, 1991). In addition, satellite cells express some molecules that are common to all peripheral glia. The molecular phenotype of satellite cells is regulated, often negatively, by factors present in the microenvironment. Schwann cell myelinating protein (SMP) (see below), a molecule expressed by both myelinating and non-myelinating Schwann cells, is expressed in satellite cells of embryonic but not adult DRG. When cultured in vitro and released from the tightly-packed microenvironment of ganglion, the satellite cells rapidly express SMP, suggesting that constitutive expression of SMP is inhibited in the DRG (Cameron-Curry et al., 1993). Krox-20, a transcription factor important for myelination, is another molecule that is down-regulated by the microenvironment of ganglion (Murphy et al., 1996). The ability of satellite cells to re-express Krox-20 only occurs after the time in development when transition of precursors to Schwann cells occurs, suggesting that the maturation programme in the satellite cells is on same time scale as that in Schwann cells in peripheral nerves (Murphy et al., 1996). Minor differences exist between the satellite cells in the sensory ganglion and those in the sympathetic ganglion. Satellite phenotypes were further regulated by difference in DRG and sympathetic ganglia, but again these differences are reversible when cells are cultured in vitro (Riidel and Rohrer, 1992).

Perisynaptic Schwann cells (terminal Schwann cells)

16 Perisynaptic Schwann cells are specialised non-myelinating Schwann cells that cover a single large terminal axon and its branches at the highly specialised synaptic zone. The outline of the elliptical soma of the perisynaptic Schwann cells can be visualised within the endplate when the nerve terminals are labelled with a fluorescent agent that has high affinity for the axolemma, for example, fluorescent peanut agglutinin (PNA) (Ko, 1987). This type of Schwann cell is different from other Schwann cells and behaves in some way more like astrocytes. Unlike non-myelinating Schwann cells, they normally do not express growth associated protein 43 (GAP-43) (Curtis et al., 1992; W oolf et al., 1992). After nerve transection, perisynaptic Schwann cells respond to denervation by up-regulating GAP-43 in 18 hours, while for myelinating Schwann cells it often takes several weeks (Tetzleff et al, 1989; Curtis et al., 1992). Glial fibrillary acidic protein (GFAP) level is also up-regulated in perisynaptic Schwann cells during denervation, utilizing a mechanism that involves blockage of neurotransmitter rather than the loss of axonal contact in the myelinating Schwann cells (Georgiou et al., 1994; lessen et al., 1990). Both myelinating and non­ myelinating Schwann cells proliferate after denervation while perisynaptic Schwann cells do not divide (Clemence et al., 1989; Connor and McMahan, 1987). They form long processes that extend ahead of presynaptic axon terminals but retract on reinnervation (Woolf et al., 1992). In frog the perisynaptic Schwann cells respond to neurotransmitters with a release of intracellular Ca^ (Jahromi et al., 1992). These differences suggest that perisynaptic Schwann cells are specialised to respond to events occurring at synapses.

The Schwann cells Schwann cells are the ensheathing cells of peripheral nerves. In adulthood they are of two mature phenotypes: Myelinating and non-myelinating Schwann cells. The former forms a lipid rich, compact multilayer membrane sheath (myelin) round the axons that are one micrometer in diameter or larger, and hence help the to accelerate the propagation of the nerve impulse. Myelin is not present or is very scarce around the ganglion cells of the PNS. The later, the non-myelinating Schwann cells, have several small axons lying separately in its grooves and provide a physical barrier within the nerves.

17 1.2 The molecular phenotypes of Schwann cells The differentiation of myelinating Schwann ceils is marked by dramatic morphological differences that are involved in myelin formation. The myelinating cells express a whole set of highly regulated molecules to meet this task. These includes protein zero (Po), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipid protein (PLP), PMP-22 (peripheral myelin protein 22), myelin protein 2 (P2), 2’,3’,-cyclic nucleotide 3’-phosphodiesterase (CNPase) (references see bellow). Another set of molecules are specifically expressed in mature non-myelinating Schwann cells. This group of molecules includes p75, the low affinity NGF receptor (p75NGF-R), neural cell adhesion molecule (NCAM), Ll/NgCAM (neural-glia cell adhesion molecule), GFAP, two proteins recognised by rat-specific monoclonal antibodies, A5E3 and Ran-2, and GAP-43 (references see follows). Although again specifically regulated, the functions of this set of molecules, some of which are shared by CNS glia, are mostly unknown, apart from N-CAM and LI (see follows). Among molecules expressed by the two phenotypes of Schwann cells, some are not specific to the Schwann cell lineage but their expression marks important stages of the development of the Schwann cell lineage. These include SI00, vimentin, laminin, and galactocerebroside (Gal-C) (references see bellow). Some are expressed by both phenotypes and are restricted in the Schwann cell lineage. This type of molecules includes seminolipid 04, 08 and 09 (references see below), the function of which are unknown. Some transcription factors and other proteins that are crucial in myelination are described below.

1.2.1 Appearance and regulation of Schwann cell phenotypes during development Schwann cell precursors present in rat peripheral nerves at embryonic day (E) 14-15 give rise to SI00 positive embryonic Schwann cells, first seen at E l6 (lessen et al., 1994). The POU-domain transcription factor SCIP (suppressed cAMP inducible POU protein, see below) is first reported in the Schwann cell precursor at E l3 in mouse nerves, equivalent to E l5 in rat embryos (Blanchard et al., 1996). At E17 over 95% of the cells dissociated from a rat sciatic nerve are SI 00 positive (lessen et al., 1994). The transcription factor Krox-20 is expressed in the entire length of spinal nerves of E l5 mouse embryo (equivalent of E l7 rat), after the transition from precursor Schwann cell, soon after S I00 expression in the Schwann cells (Topilko et al., 1994; Murphy et al., 1996). By E l8 most Schwann cells also express the lipid antigen 04, which first appears in the S I00 positive cell population at E l6 (Mirsky et al., 1990). 04 is believed to be the sulphated form of Gal-C that appears two days later in development, but it is also possible that antibody to 04 also detects an antigen other than sulphated Gal-C (Mirsky et al., 1990). By E20 95% of the S I00 positive Schwann cells are also 0 4 positive (Mirsky et al., 1990). The expression of Gal-C is first detected around birth in the myelinating Schwann cells and is only seen in the non-myelinating Schwann cell after the maturation of this cell type, which occurs around the 3rd week after birth (lessen et al., 1985). Most of the phenotypes found in non-myelinating but not myelinating Schwann cells are also expressed by the Schwann cell precursor at E l4-15, except for GFAP and S-100, which is expressed in all Schwann cells (lessen et al., 1990). GFAP expression is first detected during development at E l8 in cells that become myelinating Schwann cells as well as those that will become non-myelinating (Jessen et al., 1990). After birth the phenotypes of the presumably homogenous immature Schwann cells undergo dramatic changes depending on the type of axons they associate with. The development of myelinating Schwann cells in the nerve involves an initial up-regulation of the myelin proteins, for example, the induction of Po, MAG, MBP, and P2. This is followed by the down- regulation of molecules associated with the non-myelinating phenotype, including GFAP, N-CAM, A5E3 and p75NGF-R (Jessen et al., 1990; Jessen et al., 1987b). In the adult rat, all these down-regulated proteins are essentially undetectable by immunohistochemical methods in myelinating Schwann cells. The phenotypes of non-myelinating Schwann cells remain mostly similar to the more immature phenotypes, except for expressing Gal-C 3 weeks after birth (Jessen et a., 1985; Eccleston et al., 1987b).

1.2.2 Non-myelinating phenotypes Non-myelinating Schwann cells of adult rat express a different set of proteins from myelinating Schwann cells. These include p75NGF-R, NCAM, Ll/NgCAM, GFAP,

19 two proteins recognised by rat-specific monoclonal antibodies, A5E3 and Ran-2, and GAP-43 (Yen and Field, 1981; Jessen and Mirsky, 1984; Jessen et al., 1984; Ni eke and Schachner, 1985; Mirsky et al., 1986; Jessen et al., 1990; Stewart et al., 1992). This group of molecules tends to be regulated in the opposite direction to the way the myelinating phenotype is regulated. These markers are down-regulated in myelinating Schwann cells after the expression of myelin proteins and remain unchanged in the non-myelinating cells. p75NGF-R are down-regulated by treatments elevating the intracellular cAMP level (Mokuno et al., 1988; Lemke and Chao, 1988 Morgan et al., 1991). p75NGF-R, NCAM, LI, GAP-43, GFAP and A5E3, are up-regulated during denervation (Curtis et al., 1992; Daniloff et al., 1986; Jessen et al., 1987b; Martini and Schachner, 1988; Taniuchi et al., 1986, 1988). The maintenance or maturation of the non-myelinating phenotypes is believed to be by a default pathway, but recent results suggest that additional signals, probably regulated by TGF-(3, may be needed for the expression of the full non-myelinating phenotype. In vitro TGF-P is shown to up-regulate the expression of LI and NCAM, besides blocking cAMP-induced expression of Po and the lipid antigens Gal-C and 04 (Mews and Meyer, 1993; Morgan et al., 1994; Stewart et al., 1995). Much less known is about the significance of the phenotype of non-myelinating Schwann cells than about the myelin proteins. Being adhesion molecules, NCAM and LI may be involved in axon Schwann cell interactions at the premyelination stage, as well as in mature unmyelinated fibres (Seilheimer and Schachner, 1988; Martini and Schachner, 1986; Wood et al., 1990). p75NGF-R probably plays a role in grabbing the nerve growth factor (NGF) on the Schwann cell surface, making it available for uptake and transport by the axons during axonal growth (Johnson et al., 1988).

1.2.3 Myelinating phenotypes Po (see below)

MBP Myelin basic proteins comprise a small group of soluble, highly charged proteins, ranging in size from 14-22 kDa, whose mRNAs originate from a single gene by alternative splicing of the primary transcript (de Ferra et al., 1985; Takahashi et al.,

20 1985; Newman et al., 1987). They are a common participant in all myelin sheaths, even in the most primitive vertebrates (Saavedra et al., 1989). Different upstream regulatory sequences of MBP in the CNS and PNS have been identified (Gow et al., 1992). Both oligodendrocytes and myelinating Schwann cells target MBP proteins to myelin via translocation of MBP mRNA to the myelin sheath (Colman et al., 1982; Trapp et al., 1987; Ainger et al., 1993). A role for MBP in the structure and compaction of the major dense line of the myelin sheath was shown by the phenotypes of two dysmyelinating mice lines with a mutated MBP gene (Privât et al., 1979; Popko et al, 1988; Mol ineaux et al., 1986). In the shiverer and shiverer myelin- deficient mice, myelin is scarce in the CNS, the residual sheaths are not compacted at the major dense line, although PNS myelin appears almost normal, probably compensated for by Po (Martini et al., 1995a). When MBP is introduced by transgenic techniques into a shiverer mouse, a normal sheath is restored (Readhead et al., 1987; Kimura et al., 1989). A regulatory role in myelination for MBP is also proposed. It is known that exon II containing MBP (MBPexII) isoforms distributes throughout cytoplasm and often in the nucleus. It is now known that MBPexII is actively targeted to the nucleus and its peptide segment provides a nuclear localisation signal and probably also regulates the segregation of MBP mRNA to the cell processes (Pedraza et al., 1997).

MAG MAG is transmembrane protein that is highly glycosylated in the extracellular domain and shares homology to the immunoglobulin gene superfamily. The intracellular domain is the site of difference between the two MAG forms. The two isoforms are generated by alternative splicing: The 67 kDa form predominates in Schwann cells while the 72 kDa form is expressed in both CNS and PNS (reviewed by Hudson, 1990). In the PNS MAG is only located in the mesaxons, the Schimidt-Lantermann incisures and the paranodal loops. This restricted expression and exclusion from the myelin is related to its function of maintaining the periaxonal space and preventing compaction at the Schmidt-Lantermann incisures and the paranodal loops (Trapp et al., 1984; Trapp and Quarles, 1984). The Ig-like domain of MAG is probably responsible for its adhesive property. Apart from the nodal area, MAG is believed to

21 have roles in initiating enwrapment and promoting neurite extension in early myelination (Johnson et al., 1989). The presence of 72 IcDa form of MAG in the large multivesicular bodies suggests another possible function for MAG in retrograde transport of membrane components from the periaxonal space to the oligodendrocyte cell body (reviewed by Hudson 1990). No natural mutant for MAG has been described to date, although abnormal distribution of MAG in quaking mice, which is not mutated in the MAG gene, has been reported (Trapp et al., 1988). MAG inhibits neurite extension from older neurites via interaction with a sialoglycoprotein in culture, but not for neurites from newborn DRG neurons (DeBellard et al., 1996; Tang et al., 1997). A study of MAG knockout mice showed that regeneration is better in mice lacking MAG, supporting an inhibitory role for MAG in vivo (Filbin, 1995).

PLP Myelin proteolipid protein is the most abundant protein in CNS myelin, constituting 50% of the myelin membrane proteins in the CNS (Lees and Bizzozero, 1992). DM- 20, an alternative spliced product of PLP-mRNA precursor, is a minor component of mature myelin but is the dominant isoform during embryonic development (Schindler et al., 1990; Timsit et al., 1992, 1995). The 30 kDa PLP is a transmembrane protein with four transmembrane domain and a large extracellular domain (Milner et al., 1985; Popot et al., 1991). The primary structure of PLP/DM-20 is highly conserved across various species (Yan et al., 1993). Severe dysmyelination observed in animals whose PLP gene is mutated by a single nucleotide suggests a crucial role of this gene in myelination in the CNS (Hodes et al., 1993; Griffiths et al., 1995), probably by promoting the apposition of extracellular surfaces of the myelin lamellae (Boison et al., 1995). Its role as an ionophore has been suggested, based on its molecular topology (Helynck et al., 1983). Several human mutations are known giving rise to Pelizaeus-Merzbacher disease (type 1-3) and the X-linked complicated spastic paraplegia (SPG) (Seitelberger et al., 1995; Harding et al., 1995; Saugier-Veber et al, 1994; Cambi et al., 1996). Normal PLP function is very sensitive to gene dosage expression, as duplication of the gene results in a similar phenotype to the effect of a mutation (Readhead et al., 1994; Kagawa et al., 1994). There are four cellular phenotypes of PLP mutation (1) intracellular retention of misfolded PLP, (2) the

22 dysmyelination of CNS axons in white matter tracts (3) abnormal myelin compaction (4) premature death of oligodendrocytes (summarised in Klugmann et al., 1997). In the PNS the PLP/DM-20 is a minor component, constituting less than 1% of total myelin protein (Lemke, 1993). PLP has recently been identified in PNS compact myelin, and the new discovery of a null allele of PLP with PNS demylination demonstrates its role in proper myelin function in the PNS as well as in the CNS (Garbern et al., 1997).

PMP-22/ gas/CD25/SR13/PASII PMP-22 is a small, extremely hydrophobic integral membrane glycoprotein. Its name was given because of its abundance in peripheral myelin (about 5%) and its apparent molecular weight of 22 kDa (reviewed by Suter and Snipes, 1995). It has no apparent homology to the other identified protein sequences, but its four membrane associated domains resemble the structure of PLP and the connexin subunit of gap junctions (Lemke, 1993). The same regulation pattern of PMP-22 during axonal injury to other major PNS myelin suggests that this gene is similarly regulated by axonal contact (Suter and Snipes, 1995) This protein has attracted much attention over the past few years because mutations in the PMP-22 gene are likely to account for a set of dominantly inherited peripheral neuropathies, including Trembler (Tr) and Trembler- J (Tr-J) mice and Charcot-Marie-Tooth (CMT) disease subtype 1 A, a human neuropathy, Dejerine-Sottas syndrome (DSS), another peripheral neuropathy, and hereditary neuropathy with liability to pressure palsies (HNPP) (Timmerman et al., 1992; reviewed in Snipes et al., 1992). The integrity of PNS myelin is sensitive to level of PMP-22 (Sereda et al., 1996; Chance et al., 1993), and the difference in the nature of disorder is caused by several different mutational mechanisms affecting the PMP-22 gene (Suter and Snipes, 1995). For example Tr and Tr-J mice, which carry missense mutations in the putative transmembrane domain of PMP-22 (Suter et al., 1992 a, b) and DSS patients and some CTMT1A families in human with missense mutations in the putative transmembrane regions share similar phenotypes of severe myelin deficiency (Henry and Sidman, 1988; Valenti;n et al., 1992; Roa et al., 1993 a-c). While the vast majority of CMTl A patients carry a duplication that includes the entire PMP-22 gene (Lupski et al., 1991; Raeymackers et al., 1991), which leads to

23 slowly progressive distal muscle atrophy and weakness starting from second or third decade, heterozygous deletion of the same chromosomal region leads to HNPP (Chance et al., 1993) that is characterized by repeated weakness which is caused by pressure or trauma to affected nerves.

Genes encoding putative myelin assembly proteins; P 2 , CNPase

P 2 , a 14.8 kDa basic protein, is a minor component of peripheral myelin, accounting for only 0.05%-l% of total myelin protein. It is only present in compact myelin and not in the Schmidt-Lantermann incisures (Hahn et al., 1987). It is speculated that this protein is involved in the synthesis and transport of long chain fatty acids (reviewed by Hudson 1990). Another protein thought to be involved in the assembly of myelin is CNPase (2’,3’-cyclic nucleotide 3’-phosphodiesterase). CNPase is a cytoplasmic protein expressed in glial cells and some non-neuronal tissues as well. The 2 isoforms are 46 and 48 kDa. Developmental expression of the CNPase gene during peripheral nerve development, unlike in the CNS, does not correlate with myelin formation (Stahl et al., 1990). CNPase in the CNS may take part in intercellular transportation of mRNA, guiding specific proteins to their site of myelin assembly (Hudson, 1990)

Periaxin Periaxin is a novel protein that is only expressed in myelinating Schwann cells. It was first identified as a Triton-X-100 insoluble, 170 kDa protein. Although as abundant in the PNS myelin as MBP, CNP and MAG, it was identified relatively recently because it is prone to proteolysis. The murine periaxin gene spans 20.6 kilobases and encodes two mRNA of 4.6 and 5.2 kilobases, generated by differently sized 3’ untranslated regions, that encode two periaxin isoforms, L-periaxin and S-periaxin of 147 and 16 kDa respectively (Gillespie et al., 1994; Dytrych et al., 1998). The deduced primary structure from the sequence suggest that this protein has a large number of phosphorylation sites. A repeat of a pentamer through a long region separates the basic and acidic regions of the protein. Both proteins possess a PDZ domain at the N terminus and are targeted differently in Schwann cells. The larger periaxin is localized to the plasma membrane of myelinating Schwann cells while the S-periaxin

24 is expressed diffusely in the cytoplasm (Dytrych et al., 1998). In rat the expression of periaxin starts at birth and peak at 8 to 20 days after biith, which is a period of active myelination. Like MAG, in compacted myelin it is only expressed in the periaxonal sheath, and is believed to be involved in initial enwrapment of the axon and in the periaxonal region of the Schwann cell membrane (Gillespie et al., 1994).

1.2.4 Molecules expressed by both phenotypes SlOO Calcium-binding protein SlOO is expressed by both non-myelin and myelinating cells from several different species in vivo and in vitro. Beside the nervous system, it is also present in melanocytes, some CNS glial cells and some neurons. Its prevailing expression in all Schwann cells in mature nerves, in culture, in denervated nerve trunks and by all embryonic Schwann cells except for the Schwann cell precursor, but not in fibroblast or perineurial cells, makes it a good operational marker for cells of Schwann cell lineage (reviewed by Mirsky and lessen, 1990). The expression of SlOO in cells of Schwann cell lineage marks the transition from precursor cells to the Schwann cells, which in rat happens in the sciatic nerves between E l5 and E17 (lessen et al., 1994). The induction of SlOO, unlike many other Schwann cell phenotypes, is irreversible once triggered.

Gal-C Gal-C is a simple glycosphingolipid and is the major glycolipid of the myelin sheath. It is expressed by oligodendrocytes in the CNS and both myelinating and non­ myelinating Schwann cells in PNS (Mirsky et al., 1980; Ranscht et al., 1982; lessen et al., 1985). Gal-C is a reliable marker for oligodendrocytes in that the expression is sustained even when the oligodendrocytes are cultured in vitro (Mirsky et al., 1980). In contrast to the expression in CNS, the Gal-C expression in Schwann cells is regulated by axonal contact. When axonal contact is lost during denervation or on plating in the culture dish, Gal-C gradually disappears during a course of 6 weeks in the myelinating Schwann cells and 1 week in the non-myelinating cells in vivo, but over several days in culture (lessen et al., 1987a) During development the time course of its expression is differently regulated in the two phenotypes. It is first

25 detected around birth in the myelinating Schwann cells, and in the non-myelinating cells it is only detected after the axons were separately located within different troughs of the Schwann cell cytoplasm, the final stage of morphologenesis of the unmyelinated fibre (Jessen et al., 1985; Eccleston et al., 1987b).

04 The antigen which 04 monoclonal antibody recognizes is the sulphated form of Gal- C and perhaps some other unidentified lipids as well (Bansal and Pfeiffer, 1987). The 04 epitope is expressed two days earlier than Gal-C during development of both oligodendrocytes and Schwann cells, while sulfatide is synthesised from Gal-C using the enzyme galactosyl sulfotransferase (Sommer and Schachner, 1981; Wolswijk and Noble, 1989; Mirsky et al., 1990). It is possible that 04 recognises epitopes other than sulfated Gal-C in Schwann cells (Mirsky et al., 1990). Nonetheless, the antigen which 04 antibody recognizes is regulated by axonal signals and elevation of cAMP in a way similar to that of Gal-C (Mirsky et al., 1990). During development, the expression of 04 antigen is first detected in rat El 6-17, and by El 8 most of the cells dissociated from the sciatic nerves are 04 positive, a stage coinciding with the peak proliferation of embryonic Schwann cells (Mirsky et al., 1990). Sulfatides were shown to bind selectively to Schwann cell basal lamina (Roberts et al., 1985). This up-regulation of 04 may be related to basal lamina production (Mirsky et al., 1990).

1.2.5 Molecules not specific to Schwann cells but important in PNS myelination SCIP (Oct-6/Tst-l) The transcription factor SCIP is a member of the POU domain family, members of which are distinguished by the presence of a DNA-binding domain consisting of a homeobox and an upstream POU domain (Herr et al., 1988). The POU domain generally binds to an octamer motif to activate transcription (He et al., 1991; Meijer et al., 1992; Monuki et al., 1990, 1993). SCIP has been isolated, sequenced and analysed (Monuki et al., 1989,1990; Kuhn et al, 1991). The expression of SCIP is not restricted to PNS glia, although it is implicated in myelination (see below). SCIP is expressed in PNS and CNS myelinating cells (Monuki et al., 1989; Susuki et al., 1990; Collarini et al., 1991, 1992; Scherer et al., 1994; Zwart et al., 1996), in

26 specific populations of neurons during development (Lemke, 1993) and in neonatal testes and epidermis (He et al., 1989). In the Schwann cell lineage, the earliest expression reported is in Schwann cell precursors (Blanchard et al., 1996) and it peaks at one day after birth (Monuki, 1989). Purified Schwann cells in vitro do not express SCIP, but mRNA can be induced by elevation of cytoplasmic cAMP (Monuki et al., 1989).

Like the myelin proteins, SCIP expression is closely regulated by axonal contact. A high level of SCIP mRNA was detected during development and regeneration, and axotomy results in rapid down-regulation of this gene (Scherer et al., 1994). That SCIP expression is well in advance of myelination, and in transient assays the SCIP protein binds to the promoter of the gene encoding Po to repress its transcription (He et al., 1991), suggests a role for SCIP during myelinalion. Indeed the importance of SCIP in the enwrapping of the axon is demonstrated by several mutations in which SCIP function is defective. The implication from observation of these mutations, however, is not consistent. Weinstein et al. (1995) designed transgenic mice in which a dominant negative antagonist of SCIP prevents normal SCIP DNA binding. This antagonist was expressed under a Po promoter to be specific to the Schwann cells. This results in premature myelination, over expression of myelin specific gene products, and hypermyelination. On the other hand, null mutation of SCIP results in a constraint or delay of myelination (Bermingham et al., 1996; Jaegle et al., 1996), suggesting that SCIP is important in initiating the myelination process.

Krox-20 Krox-20, originally isolated as an immediate-early seium response gene, encodes a transcription factor with a DNA binding domain consisting of three C 2 H 2 -type zinc fingers. Krox-20 protein binds DNA in a sequence-specific manner, and it was shown to be able to transactivate a promoter next to multiple Krox-20-binding sites (Chavrier et al., 1988, 1990; Vesque and Charnay, 1992). In adult mice a 3.2 kb Krox-20 transcript is detected in the thymus, spleen and testis (Chavrier et a l, 1988). During development it is expressed in a distinct pattern in the hindbrain, being restricted to rhombomere 3 and 5, as well as in early neural crest cells, and in neural

27 crest-derived boundary cap cells and glial cells of cranial and spinal nerve and ganglion (Wilkinson et al., 1989; Herdegen et al., 1993; Topilko et al., 1994). The expression of Krox-20 is important in maintenance of rhombomere 3 and 5. The hind brain of mice homozygous for the Krox-20 mutation are disrupted in that rhombomeres 3 and 5 are completely eliminated (Schneider-Maunoury et al., 1993, 1997). Furthermore, Krox-20 is suggested to be a key regulator of rhombomere- specific gene expression in the developing hindbrain (Sham et al., 1993; Nonchev et al., 1996; Seitanidon et al, 1997). It can interact with multiple binding sites in the promoter region of several Hox genes and can induce ectopic expression of the Hox genes in vivo (Sham et a l, 1993; Nonchev et al, 1996; Seitanidon et al, 1997). Krox-20 is also an important regulator during the maturation of peripheral glia and in the process of myelination (Topiko et al, 1994; Murphy et al., 1996). The expression of Krox-20 in PNS coincides with the acquisition of maturation, demarcated by the immunoreactivity of SlOO, after the transition from the Schwann cell precursor (Murphy et al, 1996). The expression of Krox-20 in the Schwann cell lineage is regulated by axonal contact and cAMP elevation in a way similar to that of the myelinating proteins but it is also up-regulated by neuregulins (Murphy et al., 1996). The observation that myelination is prevented and myelin proteins Po and MBP are absent from myelin in Krox-20 knock out mice demonstrates the importance of Krox- 20 in regulating the expression and assembly of proteins necessary for myelination (Topilko et al., 1994).

QKI proteins Quaking viable mouse (qk^) is an autosomal recessive mutation in mice characterized by severe dysmyelination of the CNS (Sidman et al, 1964). The primary defect in qk^ that leads to the demyelinating phenotype has not been identified. Recently a new gene q k l has been identified 1.1 kb central to the deletion in the q k'’ mutation on mouse chromosome 17 (Ebersole et al., 1992, 1996). Of the known three transcripts, QKI 6 and QKl-7 are reduced dramatically in myelinating cells, whereas QKI-5 is reduced only in severely affected regions of the brain. Furthermore, abnormal expression of QKI proteins is specific to qk"’ mutants, suggesting that mutation of QKI genes contribute to the mehanisms for demyelination in the qk^ mutant. The

28 correlation of the QKI expression and qk^ mutants also suggests that the QKI proteins are regulators of myelination (Hardy et al., 1996).The three mRNAs transcribed from this gene, 5, 6 , and 7 kb in length respectively, differ only in their 3’ end and 3’ untranslated regions. The predicted amino acid sequences of this mRNA contain a KH domain, which is believed to be associated with regulation of cellular RNA metabolism (Musco et al., 1996). The QKI proteins are not restricted to the nervous system. They are also expressed in heart, lung, testis, and during development in the neuroectodermal cells of the neural tube (Ebersole et al., 1996). In the nervous system, they are normally expressed in both the glial cells of CNS and PNS. The distribution of QKI 6 and 7 are identical. The QKI proteins are present in the myelinating cells of CNS and PNS, with QKI 5 mostly in the nucleus and QKI 6 and 7 in the cytoplasm. They are also expressed in astrocytes and non-myelinating Schwann cells (Hardy et al., 1996).

Connexin 32 Connexin 32 was originally found as a liver gap junction protein (Paul, 1986), and was only recently identified as a myelin-related protein in the PNS and CNS (Scherer et al., 1995). In the peripheral nerve, it is believed to form gap junctions that shorten the communication pathway between the periaxonal cytoplasm and the peripheral cytoplasm in which the cell nucleus is located (Scherer et al., 1995). Molecular genetic studies have shown that gene mutation of connexin 32 (Cx32), a gap junction protein, results in the PNS abnormality, X-linked Charot-Marie-Tooth disease (CMTX) (Bergoffen et al., 1993; Bone et al, 1995; Bruzzone et al., 1994; Fairweather et al., 1994; lonasescu et al., 1994). Although it is expressed in many tissues, tissues other than the PNS appear to be normal (Bergoffen et al., 1993). The relationship between loss of connexin 32 function and CMTX disease is still unclear. Reduced junction permeability caused by decrease in either pore size or open channel probability, resulting in reduced permeation of second messengers such as cAMP through gap junctions, probably causes the demyelination and axonal degeneration (Oh et al., 1997).

1.2.6 Ion channels

29 Mammalian Schwann cells in culture express at least six kinds of voltage-dependent ion channels, and others may not have been detected yet. Both voltage-dependent sodium and potassium currents have been recorded using whole-cell patch clamp technique (Chiu et al., 1984). Three other voltage-dependent channels were detected when measuring the outward current: a chloride channel and two types of potassium current (Howe and Ritchie, 1988). A calcium-dependent cation-selective channel and anion selective channels have also been detected in Schwann cells (Bevan et al., 1984; Gray et al., 1984). Glial ion channels are generally similar to their neuronal counterparts, except for some sodium channels. (Mi et al., 1995; Barres et al., 1990). Using whole-cell patch-clamp recording, Schwann cells in culture also express voltage-gated potassium and sodium currents. These currents occurs at lower density than in many neurons, but are qualitatively similar to neuronal currents (Shrager et al., 1985). The expression of sodium and potassium channels are different in Schwann cells of two phenotypes, in vitro and when Schwann cells were acutely dissociated. Outward sodium channels and potassium currents have been recorded in non-myelinating Schwann cells but not in myelinating cells. However, inwardly rectifying potassium currents were found in both phenotypes (Chiu 1987, 1988). Transection of nerves results in the appearance of sodium channels in myelinating Schwann cells but down regulated the sodium channels in the non-myelinating cells, showing another example of axonal regulation of Schwann cell phenotype (Chiu, 1988).

It has been suggested that glial ion channels play a role in regulating the extracellular potassium. A rapid release of accumulated potassium from neuron to extracellular space happens whenever there is neuronal activity. It has been demonstrated that elevation of intracellular potassium in glial cells and depression in neurons occurs during neuronal firing. It has also been shown that these potassium homeostatic mechanisms involve passive fluxes of ions through channels instead of active transport of ions by pumps or extracellular diffusion (reviewed by Barres et al., 1990). Some experimental evidence supports another mechanism that allows a greater capacity to absorb extracellular potassium. More potassium could be transported and stored in the glia when inwardly rectifying potassium channels were coupled with a

30 compensatory chloride channel (Coles et a l, 1989; Barres et al, 1988; Ballanyi et al, 1987).

1.3 Neuronal influences on Schwann cells Different aspects of glial cell differentiation have been shown to be modulated by interactions with neuronal element, i.e., the neuronal body and the axons (for a review see Reynolds and Woolf, 1993; Mirsky and Jessen, 1998). Broadly speaking, neurons exert four classes of action on Schwann cells: survival, stimulation of differentiation, inducing or repressing proliferation; and modifying the migration/growth of the Schwann cells. Neuronal factors exert an important influence on Schwann cells during several developmental stages. During early development of the lineage,

Schwann cell precursors (see 1. 1 1 ) survive if cultured in the presence of neuron- derived factors, which in term promote the maturation and proliferation of precursor to Schwann cells (Jessen et al., 1994; Dong et al., 1995). The dependence on axons for survival gradually becomes smaller during development, and adult Schwann cells are independent of neuronal factor for survival, but low levels of neuron secreted factors are needed to prevent immature Schwann cells and teloglia undergoing apoptotic death (Grinspan et al., 1996; Syroid et al., 1996). Later in postnatal development two types of axons direct the differentiation of immature Schwann cells into two phenotypes in a highly ordered fashion (Jessen et al., 1990; see following section). Upon denervation, the Schwann cells respond to the mitogenic axolemma and proliferate (Salzer et al., 1980; Ratner et al., 1988). Several of the neuronal related actions on Schwann cells are regulated by neuregulin and are discussed below.

1.3.1 Regulation of the lineage choice and embryonic development of the Schwann cell lineage: signaling by (3-neuregulin The neuregulin gene encodes various isoforms which are known as NDF (neu differentiation factor; Wen et al., 1992), heregulin (Holmes et al., 1992), GGF (glial growth factor; Marchionni et al., 1993), ARIA (acelyl choline receptor induciing activity; Falla et al., 1993) or SMDF (sensory and motor neuron-derived factor; Ho et al., 1995). These EGF-like domain containing molecules have been shown to be important in the growth and differentiation of diverse cell types, among them glial.

31 muscle and epithelial cells (for reviews see Peles and Yarden, 1993; Carraway and Bruden, 1995; Marchionni, 1995; Lemke, 1996). Neuregulins modulate the cell response via binding to tyrosine kinase receptors of the erbB family. Neuregulin binds to erbB3 and erbB4 with high affinity, but not the erbB2 (HER2) and erbBl (EGF) receptors (Peles et al., 1992, 1993; Plowman et al., 1993; Carraway et al., 1994). However, erbB2 is capable of forming heterodimers with erbB3 or erbB4 and by receptor cross-phosphorylation initiates downstream signalling within the cell (Holmes et al., 1992; Wen et al., 1992; Carraway and Cantley, 1994; Beerli et al., 1995).

Experiments in cell culture and mutational analysis in mice demonstrate that the action of neuregulin, especially its interaction with ErbB3, is important for differentiation of the neural crest and Schwann cell precursors (Shah et al., 1994; Meyer and Birchmeier, 1995; Dong et al., 1995; Kramer et al., 1996; Riethmacher et al., 1997). While many growth factors are known to promote neuronal differentiation of the neural crest population, neuregulin is the only known growth factor that has an effect on the differentiation of the glial lineage from the neural crest population. Shah and colleagues (1994) showed that neuregulin inhibits neuronal differentiation and biases crest cells towards a glial cell fate in in vitro cultures of multipotent rat neural crest stem cells. Neuregulin gives an instructive signal that influences lineage determination rather than promoting glial enrichment, selective survival or proliferation. In neural crest cultures, neuregulin is expressed by neurons, suggesting that early neuroblasts might regulate the generation of glia by expressing this growth factor (Shah et al, 1994). Schwann cell precursors, a transitional stage between the neural crest and the mature Schwann cell, also depend largely on neuregulin for survival and maturation. Several lines of evidence suggest that the survival and maturation of Schwann cell precursors depends on axon-derived signals. Conditioned medium from postnatal day 1 (PI) DRG neurons supported survival of Schwann cell precursors in vitro and allowed them to convert to Schwann cells on schedule (Dong et al., 1995). Schwann cell precursors also survived when they were cocultured with DRG neurons, or were exposed to axonal membranes isolated from cultured DRG neurons (Ratner, Mirsky and Jessen, unpublished). The importance of neuregulin in

32 this survival and maturation of Schwann cell precursors is shown by experiments in which soluble hybrid protein that contains the extracellular domain of the erbB4 receptor, a highly specific receptor for neuregulin, prevents the axonal rescue and subsequent maturation of Schwann cell precursors (Dong et al., 1995). Several observations in vivo support the idea that P neuregulin is the neuronal derived factor that directs the development of the Schwann cell precursors. Very high level of expression is seen in both DRG and ventral horn motor neurons at E l4 and later ages in rat (Meyer and Birchmeier, 1994; Bermingham-McDonagh et al., 1997; Marchionni et al., 1993) Very few Schwann cell precursors were seen in the ventral root and growing emhi yonic nerves in mice deficient of neuregulin or its receptor, ErbB3, again suggesting that neuregulin plays an important role in the survival of Schwann cell precursors (Meyer and Birchmeier, 1995; Riethmacher et al., 1997). Furthermore, neuregulin stimulates postnatal Schwann cell proliferation and inhibits apopototic Schwann cell death. The axonal signal which is mitogenic to Schwann cells is probably regulated by membrane-bound neuregulin and Schwann cell associated ErbB2 an i ErbB3 (Morrissey et al., 1995; Reithmacher et al., 1997). Neuregulin rescues Schwann cells from developmentally regulated programmed cell death (Syroid et al., f^96). P neuregulin was also reported to promote Schwann cell migration and neuritc outgrowth (Mahanthappa et al., 1996; Calle, Mirsky, Jessen, unpublished), which might be important during the initial interaction between cells of the Schwann cell lineage and the growing neurites.

1.3.2 Axonal regulation of Schwann cell phenotypes The processes by which immature Schwann cells differentiate into the mature phenotypes are delicately regulated to meet the functions needed in the two different types of axons. It has been long established that the Schwann cell differentiation is driven by cell-extrinsic signals, most of which come from axons. If removed from the axons before differentiation by culturing in vitro or denervation, immature Schwann cells fail to differentiai(' into either of the mature phenotypes (Mirsky et al., 1980; Lemke and Chao, l9Fh) Even in the mature animal, the Schwann cell phenotypes are very liable to change if not supported by axonal contact. If a mature nerve is transected, the myelinating and non-myelinating cells in the distal stump will

33 promptly undergo numerous and radical changes in morphology and gene expression which result in a developmentally regressed phenotype. For the myelinating Schwann cells, the loss of axonnI contact results in loss of myelin markers and an expression of an immature phenotype that is very similar to the mature non-myelinating phenotype. This reversion of the phenotypes can be seen when cells are dissociated from the adult nerve and are plater! in culture without neurons, or transplanted to a nerve environment which will induce myelination in Schwann cells (Aguayo et al., 1972; Weinberg and Spencer, 1976; Trapp et al., 1988; Mirsky et al., 1980; Lemke and Chao, 1988; Jessen & Mirsky 1991), Apart from Schwann cells, sub-types of PNS glia are also known lo depend on continuous axonal contact to maintain their phenotypes. For example, the phenotype of teloglia is regulated by neurotransmitter released by presynaptic axons (Jahomi et al., 1992), and some myelin proteins are actively down-regulaieci in enteric glia and satellite cells (Dulac et al., 1990; Cameron-Curry et al.. 1993). In contrast to the molecules specific to the myelinating Schwann cells, the expression of Gal-C, 04 antigen, the lipid antigens 08 and 09, are up-regulated in both 'Ahwann cell pathways during development (Eccleston et al., 1987a). The expression of these lipids, like the proteins, is axon-dependent and is rapidly down-regulated when Schwann cells are withdrawn from axonal contact (Jessen et al., 1987a). These axonal signals, although crucial in the differentiation of Schwann cells, are still not clear. Treatments that produce elevation of intracellular cyclic AMP levels have widespread effects on the phenotypes of the Schwann cells (Mirsky et al., 1990; Morgan et al., 1991; Sobue et al., 1986). Besides synergising with growth factors to promote Schwann cell proliferation, they induce expression of 04 and Gal-C and up-iegulate the expression of myelin genes such as Po, MBP, and CNPase (Mirsky and .lessen, 1990; Lemke and Chao, 1988). The state of Schwann cells in the cell cycle, as well as the extent of maturation of the cells, seems to contribute to the choic e of programs following elevation of intracellular cAMP levels

(Mirsky et al., 1990; Morgan et al., 1991; Mirsky and Jessen, 1990).

1.4 The Wallerian degeneration system Injuries to peripheral nerves result in a sequence of specific changes in the nerve stump distal to the injury. Collectively these changes are referred to as Wallerian

34 degeneration. The reaction at the site of the injury differs depending on whether the sheath of the injured nerve fibre is severed. The scale of macrophage participation is larger, accompanied by an early inflammatory reaction, when the sheath is ruptured (Sunderland, 1978). The time course and extent of degeneration also differs depending on the type of insult, distance from the neuronal body, diameter of axon, age of animal, as well as species. In general, the axon loses action potential, fast or slow transport and neuronal transmitter secretion immediately after axotomy (Weller and Carros-Navarro, 1977; Sunderland, 1978).The onset of degeneration of the axon was reported from within 10 minutes in finer axons after mild crush, or in some cases in 48 hours (Sunderland, 1978). This is followed by a down-regulation of the myelin genes (see previous section). The first week sees breakdown of axonal component and release of axonal cytoplasm, and the myelin sheaths break up into ovoids. Over the next few weeks further degeneration includes breakdown of axonal fragments and myelin by Schwann cells and macrophages, and lysosomal enzymes digest the lipids (Sunderland, 1978). The basal lamina that surrounds each Schwann cell in the distal nerve stump remains intact and the Schwann cells remain within their basal laminae while they proliferate after axotomy (reviewed by Scherer and Salzer, 1996). This Schwann cell multiplication results in the formation of bands of Bungner.

The fragmented axonal membrane, and myelin debris were thought to contribute to the mitogenic effect on Schwann cells seen in Wallerian degeneration (Salzer et al., 1977; Salzer and Bunge 1980; Sobue and Pleasure, 1985; Bigbee et al., 1987; Clemence et al., 1989). The axonal signal might involve neuregulin signaling via its receptor ErbB2 (Morrisey et al., 1995). It has also been suggested that macrophages that have phagocytosed myelin produce a conditioned medium that is mitogenic for the Schwann cells (Baichwal et al., 1988), and indeed the peak number of macrophages present in the distal stump coincides with proliferation of non­ myelinating cells and precedes that of myelinating cells by one day (Clemence et al., 1989). However, Schwann cells can still proliferate without macrophages, though not as rapidly as when macrophages are present (Fernandez-Valle, 1995). The importance of macrophages in the phagocytosis was shown by an axotomy system using segments of nerve inside diffusion chambers which cells could not enter. The Schwann cells in

35 the chamber did not proliferate, and the myelin was discarded but not phagocytosed (Beuche and Freide, 1984; Scheldt and Friede 1987). Other roles of macrophages in the degenerating distal stump including recycling lipids from decomposed myelin by producing apolipoprotein E (Snipes et al., 1986). Furthermore, macrophages are known to produce cytokines such as interlukin-1 and interferon a and (3 (Klein, 1990).The roles of macrophages in the degenerating distal stump are still not fully understood, but is likely to involve clearing of debris and stimulation of proliferation in Schwann cells.

1.5 Myelination and the structure of myelinated fibers Schwann cells myelinate axons which have a diameter larger than IpM. In rat the process of myelination starts around birth. It is reported that the maturation of ventral roots precedes that of dorsal roots (Niebroj-Dobosz et al., 1980). Initial axon-glial contact is almost certainly mediated by adhesion molecules (Schachner, 1990). Myelination begins with enwrapping by the Schwann cells to form a jelly-roll like structure, followed by the extrusion of cytoplasm from the myelin sheath (Bunge, 1989). Tight adhesion is formed between intracellular membranes as well as opposing extracellular membranes, by the structural myelin proteins. Where the cytoplasmic faces of the membrane appear to merge the major dense line is seen in the electron microscope, and the apposition of the extracellular faces forms the intraperiod line. Active myelination is seen between one day to three weeks posnatally. The axons are myelinated by consecutive Schwann cells. The gaps between segments of sheath are called Nodes of Ranvier. In the myelinated fibres, the nerve impulse is passed down from node to node with very little loss in the amplitude of the action potential due to the low capacitance of myelin segment between the nodes. A high density of sodium channels is found at nodes of Ranvier (Ritchie and Rogart, 1977). This results in a much higher membrane current per unit area being generated at nodes than that generated in unmyelinated axons. This specialization in structure corresponds to the need to generate enough electric field at the node of Ranvier to fire the action potential in the axon membrane to (he next node. In the paranodal area, the myelin lamellae are separated at the major dense line to accommodate loops of Schwann cell cytoplasm. There is a specialised contact between axons and Schwann cells in the

36 paranodal area. The periaxonal space is reduced to 3 nm and crossed by bands or septa (reviewed by Bray et al., 1981). The functional significance of this contact might be to restrict sodium channels to the nodal region (Black et al., 1990; Salzer, 1997), to do with ion coupling between axons and Schwann cells (Barres, 1990), or to ensure electrical isolation of the nodal and internodal extracellular spaces (Livingston et al., 1973). PNS myelin differs from that of the CNS in that its inner and outer myelin lamellae are not compacted but contain thin layers of cytoplasm (Mugnaini et al., 1977). The cell cytoplasm is connected by channels called 'Schimidt-Lantermann’ incisures. This continuity of Schwann cell cytoplasm within the myelin sheath may serve as a channel to transfer proteins from the Schwann cell body, or transport phospholipids from axon to myelin (reviewed by Bray et al, 1981).

1.6 Other function of the Schwann cells Schwann cells were originally believed to be structural cells and act as an adhesion and nurturing component for the axons, but modern research suggests that they are playing active roles in the nervous system, like the neuronal component. The Schwann cells promote neuronal survival and differentiation by a series of actions (reviewed by Reynolds and Woolf, 1993). Growth factors produced by Schwann cells are known to have an important role in promoting neuronal survival and differentiation (reviewed by Scherer and Salzer, 1996; Mirsky and Jessen, 1998). The restriction of the ion channels of myelinated axons to the paranodal regions is regulated by Schwann cells. Following demyelination, sodium channel expression in the axonal membrane becomes diffuse and is restored to clusters in new nodes of Ranvier by the presence of Schwann cells during re-myelination (Dugandzija- Novakovic et al., 1995). Schwann cells also express different types of ion channels to maintain the ionic balance of axons (Beven, 1990; Barres et al., 1990). During increased nerve terminal activity, additional transmitter release could trigger changes at peri-synaptic Schwann cells that would stabilize the synapse or enhance synaptic efficacy (Georgiou et al., 1994).

1.7 The Po molecule

37 Po is an integral membrane glycoprotein of molecular weight 28 kDa. The human Po gene, MPZ, was shown to map to chromosome Iq22-q23 (Hayasaka et at., 1993) and the rat Po gene has been isolated, sequenced and analyzed (Lemke and Axel, 1985; Lemke et al., 1988). In rats and mice, this gene consists of six exons distributed over 7kb of DNA. The topographic arrangement of these exons in the genome is consistent with the functional segregation of the Po protein into extracellular, membrane- spanning, and cytoplasmic domains (Lemke et al., 1988). The primary amino acid struture of Po has been deduced from cloned cDNAs (Lemke and Axel, 1985) and bovine Po has been directly determined by protein sequencing (Sakamoto et al., 1987). It consists of a single membrane spanning region, a large extracellular hydrophobic region and small basic cytoplasmic region (Figure 1-1). Being a primitive member of the immunoblobulin-related protein family, the extracellular domain of Po is highly glycosylated (see below) and is responsible for the homophilic adhesion properties of Po and the formation of the intraperiod line of compacted myelin (Figure 1-2). In vitro the recombinant extracellular domain is capable of forming tetramers and dimers, and the same interaction is believed to play an essential role in forming a network to secure the adhesion between apposing Schwann cell membranes (Shapiro et al., 1996). Po molecules are believed to interact via electrostatic charges with the negatively charged lipid bilayer of the opposing memebrane via its basic intracellular domain in order to form the major dense line in myelin (Ding and Bruden, 1994) (Figure 1-2). Apart from the secondary structure, various post-translational modifications are also believed to contribute to Po function. The Po molecule undergoes many post-translational modifications, including glycosylation (Kitamura et al., 1976; Matthieu et al., 1975; Quarles, 1980), phosphorylation (Brunden and Poduslo, 1987b; Singh and Spritz 1976), sulphation (Matthieu et al., 1975), and acylation (Agrawal et al., 1983). Among these modifications, HNK-1-reacting glycans and mannose-rich N-glycans are potentially involved in cell adhesion (Bollensen et al., 1987; Griffith et al., 1992; Brunden, 1992).

The importance of Po in myelination is demonstrated by un compacted myelin in mice in which the Po gene is knocked out by insertionally inactivation or in Schwann cells

38 in which Po is neutralized by antisense mRNA (Owens and Boyd, 1991; Giese et al., 1992; Martini et al., I995a,b). Defects in the human myelin protein zero gene are linked to several peripheral neuropathies including Charcot-Marie-Tooth disease type IB, Dejerrine-Sottas disease, and congenital hypomyelination (Hayasaka et al., 1993; Kulkens et al., 1993; Warner et al., 1996). The adhesion properties of the extracellular domain of Po have been suggested to play a role in the initial contact of glia to the axons and in the compaction of myelin. Po is capable of homophilic binding when ectopically expressed in transfected cells (Filbin et al., 1990; D’Urso et al., 1990). It is also reported that when ectopically expressed in carcinoma cells, the host cells could revert to an epithelial phenotype (Doyle et al., 1995). When coated on plastic, Po extracellular domain promotes neurite outgrowth of DRG neurons (Schneider-Schaulies et al., 1990). Nonetheless, it is still not quite clear how the molecule achieves its adhesive ability and compaction. It is suggested that L2/HNK-1 carbohydrate, as well as protein-protein interactions, mediate homophilic binding (Griffith et al., 1992; Filbin and Tennekoon, 1991, 1993). However, the crystal structure of Po suggests that Po molecules could form a lattice and hold two opposing membranes together, even without glycosylation (Shapiro et al., 1996). No matter whether it is associated with the polypeptide chains or with the glycans, the adhesion ability of Po provides an essential force in forming and maintaining the myelin sheaths.

In adults the Po gene is believed to be expressed exclusively in myelinating Schwann cells (Trapp et al., 1981; Webster and Favilla, 1984; Hahn et al., 1987; Webster et al., 1987), and make up approximately 50% of the total myelin protein (Greenfield et al., 1973). The expression is restricted to the mid-internodal perinuclear area of Schwann cell cytoplasm, with no significant signal appearing in Schwann cells associated with unmyelinated axons (Trapp et al., 1987; Griffiths et al., 1989). In Schwann cells undergoing active myelination Po mRNA signal is found in the rough endoplasmic reticulum (RER) (Lamperth et al., 1990) and the protein in the compact myelin sheaths (Trapp et al., 1981; Trapp and Quarles, 1984; Martini et al., 1988). No Po signal is found in satellite cells of DRG in adult, although a much lower level than the myelinating one is transiently expressed during development (Lamperth et al..

39 1989). Both Po protein synthesis and mRNA levels rise about 30- to 40 fold from birth to their peak in actively myelinating nerves during the second week and fall to lower levels in adult nerves (Lemke and Axel, 1985; Trapp et al., 1988; Stahl et al., 1990; Baron et al., 1994). Schwann cells require continual positive signals from axons in order to synthesize high levels of Po (Weinberg and Spencer, 1976, Trapp et al. 1988, Mirsky et al., 1980; Lemke and Chao, 1988; Jessen & Mirsky 1991, Fernandez-Valle et al., 1993, Scherer et al., 1994, Gupta et al., 1993). In other words, the presence or absence of competent axons markedly influences the expression of the Po gene (and other myelin protein genes) in Schwann cells. Axotomy is followed by a rapid and profound down-regulation (Le Blanc et al., 1987; Gupta et al., 1988, Trapp et al., 1988). This loss of phenotype is flexible in that reinnervation is associated with re-expression (Le Blanc et al., 1987; Mitchell et al., 1990). When associated with neurites that are usually not myelinated, the Schwann cells express very low levels of Po protein (Aguayo et al., 1972; Brunden et al., 1992). Although the Po is largely down regulated when deprived of signal from axons larger than 1 |j.M in diameter, a basal level of Po mRNA in this condition has been reported by several groups (Poduslo et al, 1985; LeBlanc et al., 1987; Brunden and Poduslo, 1987; Brunden et al., 1990; Brunden, 1992; Kreider et al., 1988; Burro ni et al., 1988; LeBlanc and Poduslo et al., 1990). Very low levels of protein are detected in denervated nerves, in Schwann cells in culture, or Schwann cell co-cultured with SCG neurites (Mirsky et al., 1980; Poduslo et al., 1985; Morrison et al., 1991; Brunden et al., 1992), coincident with the presence of basal levels of Po mRNA.

The nature of the axonal signal is still unclear. Elevation of cellular cAMP levels can partially mimic the effect of axonal signals by inducing the expression of Po in neuron-free, quiescent Schwann cell cultures (Sobue and Pleasure, 1984, Lemke and Chao 1988, Monuki et al., 1989, Morgan et al., 1991, 1994; Mews and Meyer, 1993). Growth factors including IGF-1, triiodothyronine and progesterone were shown to promote Po gene expression, in the absence of serum or in very low levels of forskolin (Stewart et al., 1996; Tosic et al., 1992; Koenig et al., 1995). Growth factors of the FGF, neuregulin and TGF-(3 families inhibit the expression of Po mRNA and protein in cultured Schwann cells ( Morgan et al., 1994, Mews and

40 Meyer, 1993; Einheber, et al., 1995; Cheng and Mudge, 1996), and the effect is more profound when the cell is in the cell cycle (Morgan et al., 1994). DNA synthesis, when induced by serum or growth factors including PDGF, bFGF, TGF-p at the presence of cAMP, inhibits the cAMP induced Po level (Morgan, et al, 1991, 1994). A study of transcriptional elements of the Po gene shows that cis-acting elements reside within a 500 bp region of DNA upstream of the coding sequences region (Lemke et al., 1988; Brown and Lemke, 1997). The promoter fragment does contain a known cAMP response element (Brown and Lemke, 1997), and it alone can drive Schwann cell specific expression of heterogeneic genes (Messing, 1992, 1994). In vitro, this promoter site can be specifically bound and repressed by Tst-1, a member of the POU domain gene family (He et al., 1991)

The product of Po gene is regulated mostly at transcriptional level. Po mRNA and protein expression show a similar pattern during developement, and in denervation the down-regulation of the protein is accompanied by a down-regulation of the mRNA (Lemke and Axel, 1985; Stahl et al., 1990). Basal Po mRNA could be expressed when no protein is expressed, during denervation or when the cells are cultured without serum or ascorbic acid. (Poduslo et al, 1985; LeBlanc et al., 1987; Brunden et al., 1990; Kreider et al., 1988; Burroni et al., 1988; LeBlanc and Poduslo et al., 1990, Morrison, 1991). In other word, a translational regulation of Po gene product exists for the low level expression of mRNA, when there is minimal axonal contact. Post-translational regulation by catabolism of Po is also reported (Brunden et al., 1990). When deprived of axonal contact by denervation, posttranslational oligosaccharide processing of Po is altered, and the glycoprotein is degraded in lysosomes soon after its formation (Poduslo, 1985; Brunden and Poduslo, 1987).

The fact that Po has a single Ig domain which is encoded by two split exons and is remarkably adhesive, suggests that Po resembles an ancestral immunoglobulin gene superfamily member that would have arisen when metazoans first developed. Some Po-like molecules might, by mechanisms of gene duplication, have yielded the wide variety of immunoglobulin-like molecules seen in contemporary organisms (Colman et al., 1996). Po-like molecules are found and sequences cloned in representative

41 species of the mammalian, avian, amphibian, bony fish, and cartilaginous fish (Lemke and Axel, 1985; Sakamoto et al., 1987; Barbu, 1990; Takei and Uyemura, 1993; Stratmann and Jeserich, 1995; Saaveder et al., 1989). The overall homology of Po-like molecules in chick and trout to rat is 78% and 51%, respectively (Barbu, 1990; Stratmann and Jeserich, 1995). The Po gene is a major component of the CNS myelin in the fish but during evolution it is excluded from the CNS and replaced by PLP/DM-20 in terrestrial veterbrates (amphibia, reptilia, aves, and mammalia). Two alternatively spliced forms of trout Po mRNA species have been reported for CNS and PNS respectively, and several mRNA species for the Po gene have been reported in chick (Stratmann and Jeserich, 1995; Barbu, 1990), although other researchers suggest that the detection of several species of embryonic chick Po mRNA arises from cross reaction with 28S rRNA (Zhang et al., 1995). Although at the protein level various Po species are different in that a substantial number of substitutions are found, the overall structural similarity is retained (LeBlanc and Mezei, 1986; Barbu, 1990; Stratmann and Jeserich, 1995). Post-translational modifications seem to be conserved across species (Lemieux et al., 1995).

1.8 The neural crest The neural crest is a group of cells which emerges as the neural plate folds to form the neural tube. Before their emigration, these cells are morphologically similar to epithelium-like neural tube cells. Evidence suggestes that neural crest cells are not segregated within the tube and are equipotent to the other cells located more ventrally in the tube (Bronner-Fraser and Fraser, 1989). The premigratory neural crest cells switch their adhesion between cells to more cell-substrate interactions, and exit from the dorsal midline. After their emergence, these cells proliferate and differentiate as they migrate along defined pathways to form seemingly unrelated types of cells, which include; 1) the neurons and nonneuronal cells of the sensory, sympathetic, and parasympathetic nervous system, 2) certain endocrine and paraendocrine cells 3) melanocytes 4) skeletal and connective tissue components of the head (see table 1-1, adapted from Le Douarin and Smith, 1988; Weston, 1991)

42 Table 1-1 Derivatives of the neural crest

Classification Cell type Specific tissues or cells

PNS Neurons Sensory neurons of. Spinal ganglia Trigeminal (V) root ganglion, facial (VII) root ganglion,glossopharyngeal (IX) root ganglion, vagal (X) root (jugular ganglion) Autonomic neurons of: Sympathetic ganglia Parasympathetic ganglia Enteric ganglia Glial cells Satellite cells in: All sensory ganglia, including geniculate (VII), otic (VIII), petrosal (IX), and nodose (X) Sympathetic and parasympathetic ganglia Enteric ganglia Schwann cells of peripheral nerves Mesectodermal Skeletal tissue Nasal and orbitary skeleton derivatives Palate and maxillary skeleton Trabecule Sphenoid capsule Cranial vault: Squamosal Frontal Otic capsule Visceral skeleton Connective Dermis, smooth muscle and adipose tissue of tissue the skin in face and ventral part of neck and muscle Ciliary muscles Striated muscle of face and neck Wall of large arteries derived from aortic arches

43 (except endothelial cells ) Tooth papillae (except endothelium of blood vessels ) Corneal ‘endothelium’ and stromal fibroblasts Meninges in: Prosencephalon mesecephalon Connective tissue of: pituitary lacrymal glands salivary glands thyroid and parathyroid thymus Dorsal fm mesenchyme (amphibians and fishes) Other Endocrine and Carotid body type I cells derivatives paraendocrine Calcitonin-producing cells (C-cells) cells Adrenal medulla Melanocytes

(adapted from Le Douarin and Smith, 1988)

1.8.1 Experimental approaches Development of the neural crest has for a long time interested the embryologist. The book on this subject by Horstadius (1950) reviewed mostly the development of neural crest in amphibian embryos from the beginning of the century. Because of the nature of their incubation environment, the embryos of amphibian and avian could be operated on and observed, and they were, compared to mammals, a popular model for the study of neural crest in the pioneer studies. Among these studies were the construction of embryonic chimeras of species differing by the size or the staining properties of their cells (see Le Douarin 1982 for reference), and radioisotopic labelling of the nucleus with tritiated thymidine (Weston, 1963). The study of fate maps of the neural crest flourished with the introduction of the quail-chick marker

44 system (Le Douarin 1973). This method is based on the structural differences in the interphase nucleus in these 2 species. Explants could be taken from the donor and grafted into the same or different locations of the same or different developmental stages of the host in ovo. This is a useful tool but still was not applied to mammals. Monoclonal antibodies NC-1 and HNK-1 (Vincent et al, 1983; Abo and Balch, 1981) both recognize a carbohydrate epitope on the majority of migrating neural crest cells (Tucker et al, 1984). By examining whole mount embryo or sections through embryos stained at different stages, pathways of neural crest migration can be inferred, both in avian and murine species (Bronner-Frasier, 1986; Loring and Erickson, 1987; Erickson et al., 1989). However this approach has several pitfalls. Firsly the antibodies are not entirely specific to the neural crest population, and do not recognize all neural crest cells. Secondly only a static picture of neural crest migration could be gained this way. Some time later two other methods were adapted to follow cell lineages of single cells in vivo. One is a retrovirus-mediated gene transfer method, in which a recombinant retrovirus bearing the E. Coli P- Galactosidase (lacZ) gene infects a progenitor and is inherited by its progeny, which can then be identified by a histochemical stain for lacZ (Sanes et al., 1986; Price et al, 1987; Sanes, 1989). The other method is to inject vital dye Dil into a single cell or the lumen of the neural tube or directly into the neural folds (Serbedzija et al., 1989, 1990, 1991, 1992; Bronner-Frasier and Frasier, 1988, 1989). The dye serves to mark descendants of the injected cell until the dye fades or is diluted excessively because of cell proliferation.

1.8.2 The migrating pathways Trunk Using the quail-chick chimera system and the HNK-l/NC-1 staining method, the pathways by which neural crest cells migrate and the fate map of the derivatives was constructed (for review see Bronner-Fraser, 1993). Weston (1963), using the tritiated thymidine method, discovered two primary pathways in the trunk of chick: dorsolaterally under the ectoderm to give rise to melanocytes, and ventrally through the somite, between the sclerotome and dermamyotome, to give rise to cells in the DRG, sympathetic ganglia, adrenomedullary cells, and aortic plexuses (Figure 1-3).

45 Interestingly neural crest cells which migrate ventrally move in a segmental fashion through the rostral half of each somite, but do not move through the caudal half (Rickmann et al., 1985; Weston, 1963, Erickson et al, 1992), while those that migrate dorsally under skin do so in an unsegmented manner (Serbedzija et al., 1989, 1990, Erickson et al, 1992). This population of cells appears to migrate much later than those that take the ventral pathway (see Erickson and Goins, 1995) to give rise to melanocytes (Dusahne, 1935, Dorris, 1939, Rawles, 1947; Mayer, 1973).

Based on knowledge gained by the chick-quail chimera system and HNK-l/NC-1 staining method, apart from migrating through the two main pathways (the ventral and the dorsal lateral pathways), some cells of the neural crest population were reported to migrate A. intersomitically, B longitudinally along the neural tube, or C. logitudinally along the dorsal aorta. The intersomitic migration pathway of neural crest cells is reported using the chick-quail system (Le Douarin, 1982; Teillet et al., 1987). Erickson et al (1989) reported that the crest cells in a 28-somite chick stained with the HNK-1 antibody first collect in a wedge and begin to migrate in the intersomitic space at the 23rd somite pair, preceding the ventral migration. This intersomitic population then migrates longitudinally along the dorsal aorta and probably gives rise to the sympathetic ganglia (Loring and Erickson, 1987; Erickson et al., 1989). The longitudinal migration along the dorsal aspect of the neural tube was reported by Teillet et al (1987). When migration begins, neural crest cells leave the neural primordium in a uniform manner, and also progress longitudinally along the neural tube from anterior to posterior somitic halves, and vice versa (Teillet et al, 1987). Because of these pathways are topologically more difficult to dissect and to reconstruct, whether the cells utilizing these pathways contribute to the same population and phenotype of cells as those migrating through the main pathways, is not clear. Furthermore, reports about these migration pathways contradict each other. The timing of the intersomitic pathway reported by Erickson et al. and Teillet differs, the former suggests that intersomitic migration precedes the ventral lateral migration while the latter reported that both events occur at the same time. The evidence for migration logitudinally along the tube was indirect. These inconsistencies in these

46 reports, however, give an idea of how difficult it is to describe a highly mobile, three dimensional, heterogenic cell population using morphological evidence.

Head The cranial neural crest cells differ from the trunk crest in several ways. The cranial crest contributes to the formation of sensory and autonomic ganglia and gives rise to a range of mesenchymal tissues produced elsewhere by the mesoderm (Le Lievre and LeDouarin, 1975; Noden, 1975). The migrating pathways of the cranial crest are much more complex. The migrating cranial neural crest in mouse groups into three broad streams from the dorsal aspect of the neural tube migrating ventrally from the caudal forebrain, midbrain and hindbrain, while in chick and probably also rat only the midbrain and hindbrain give rise to neural crest (Serbedzija et al., 1992). None or very few neural crest cells were found to migrate into the otic vesicle (Le Douarin, 1982; Lumsden et al., 1991; Serbedzija, 1992). Neural crest arising from the level of forebrain migrates ventrally in a non-segmented, continuous stream through the mesenchyme between the eye and the diencephalon. The neural crest from the midbrain level migrates ventrolaterally through the mesenchyme between the surface of mesencephalon and the ectoderm (Le Douarin, 1982; Lumsden et al., 1991; Serbedzija, 1992). Cells from mesencephalic and rhomb encephalic levels migrate into their neighboring branchial arches corresponding to the segmented disposition of the rhomb encephalon. Branchial arches 1,2 and 3 receive crest cells migrating from rhombomeres 2, 4, and 6 respectively, where the cranial nerve entry/exit points reside, avoiding mesenchymal space lateral to rhombomere 3 and 5 (Lumsden et al., 1991). Enhanced levels of cell death were found in the dorsal midline of rhombormere 3 and 5, suggesting that the lack of migrating crest from these segments is probably due to elimination instead of lack of emigrating cells (Lumsden et al., 1991). The cranial crest cells populate their destination in a ventral to dorsal fashion, as is found in the trunk area (Serbedzija et al., 1992). As the result of the segmental migration of the cranial neural crest, the cranial ganglia are arranged along the the hindbrain facing the even numbered rhomb orm eres. The sensory and motor axons cross the border of the CNS and PNS via the same entry/exit point, again in the even numbered rhombomeres (Lumsden and Keynes, 1989; Golding et al., 1997).

47 1.8.3 Molecular markers for neural crest Early studies of the neural crest largely depended on the study of the HNK-l/NC-1 epitope in the chick (Bronner-Fraser, 1986; Erickson et al., 1989; Loring and Erickson, 1987; Tucker et al., 1984). However the HNK-1 antibodies are not entirely specific to the neural crest population, and do not recognize all neural crest populations. Considering that heterogenity of the population of neural crest cells exists even before and soon after emigration, it is not suprising that immunocy to chemical studies of the neural crest have identified many molecules that are expressed in various stages of neural crest emigration and only in certain sub­ populations. No single molecule to date is identified as labeling all neural crest cells. Nonetheless several molecules are expressed in sub-sets of the early migrating population of the neural crest and the combination of the information gained by the expression of these molecules gives a good indication of the migrating behavior of the neural crest. 4E9R, AP-2, CRABP, ErbB3, p75NGF-R, Sox-10, were shown to be either reliable markers for the migrating neural crest, or expression is found in most of the migrating population (Kubota et al., 1996; Mitch el, et al., 1991; Denchker et al., 1990; Maden et al., 1994; Meyer and Birchmeier, 1995; Stemple and Anderson, 1992; Kuhlbrodt et al., 1998; Southard-Smith et al., 1998). The expression of AP-2 is restricted to cranial neural crest and plays important role in cranialfacial formation (Schorle et al., 1996; Zhang et al., 1996). CRABP-1 (cellular retinoic acid binding protein 1) is involved in the retinoic acid sensitive mechanism of teratogenesis and neural development (Lyn and Giguere, 1994). ErbB3 is a receptor for neuregulin and is expressed in neural crest populations as soon as they emigrate from the neural tube (Meyer and Birchmeier, 1995). p75NGF-R is not restricted to the neural crest population and but is expressed in mesodermal tissue as well, but has been used to immunoselect neural crest stem cells in culture (Yan and Johnson, 1988; Stemple and Anderson, 1992). Sox-10 and 4E9R are recent identified molecules that are expressed with fidelity in most of the neural crest population (Kubota et al., 1996; Kuhlbrodt et al., 1998; Southard-Smith et al., 1998). Other less well clarified markers claimed to expressed in the neural crest are often only expressed after the specific fate of the cell lineage has been determined. The early markers for neuronal and glial lineages are

48 introduced in Gliogenesis and Neurogenesis During the Formation of DRG in this chapter.

1.8.4 Control of the migration pathway Factors contributing to the control of neural crest migration include the intrinsic segmental pattern of the neural tube, as well as the adhesion molecules of the extracellular matrix and possible factors secreted from the tissue which lie in the pathway of the migrating neural crest (reviewed by Bronner-Fraser, 1993; Perris, 1997). These factors also contribute to deciding the location of the entry/exit zone in the neuroepithelium, and there is a possible correlation of entry/exit zone and with the way neural crest migrates (Golding et al., 1996; Netherlander and Lumsden, 1996). Several glycoproteins have been described along neural crest migratory pathways, including laminin (Krotoski et al., 1986), fibronectin (Thiery et al., 1982), tenascin/cytotactin (Tan et al., 1987) and various collagens (Perris et al., 1991). Fibronectin and laminin attract neural crest cells to migrate in vitro (Rovasio et al., 1983; Newgreen, 1984). The migration of neural crest cells is severely disturbed when antibodies against several extracellular matrix molecules, including the beta subunit of integrins, fibronectin, laminin-heparan sulphate-proteoglycan complex, tenascin and galactosyltransferase, were injected into the heads of early chick embryos, suggesting their role in facilitating the migration of head neural crest cells (Erickson and Perris 1993; Bronner-Frasier, 1985). In the trunk the neural crest is known to migrate only through the anterior half of the somite soon after initial emigration (Weston, 1963; Rickman et al., 1985). When the anterior-posterior axis of the somite is rotated 180^ degrees the restricted migration of neural crest, as well as the motor exit point, reverts to the posterior (Bronner-Fraser and Stern, 1991). Furthermore, giant ganglia were produced when multiple anterior somites were transplanted (Kalcheim and Teillet, 1989), demonstrating that the segmental pattern is governed by the adjacent somites. The rather non-specific expression of permissive molecules in vivo argues against a hypothesis that these adhesion molecules pave the road for the neural crest, and evidence suggest a hypothesis that inhibitory factors in the extracellular matrix of the posterior part of the somites contribute to the restricted migration (Tan et al., 1987; Krull et al., 1995; Lan dolt et al., 1995). Structures which

49 regulate the dors al-ventral movement of the neural crest include the neural tube, otic vesicle, and notochord. Transposition of neural tube and notochord grafts before neural crest emigration results in crest cells that migrate following the new environment (Stern et al, 1991), possibly regulated by the expression of some embryonic form of chondroitin sulfate proteoglycan (Kerr and Newgreen, 1997).

1.8.5 The plasticity and the heterogeneity of the neural crest population It has been demonstrated that neural crest cells are undiferentiated at the time of emigration and that environmental influences are important in directing the lineage specification of phenotypes among the crest cells, using the quail-chick chimera system, when fragments of the nueral crest are heterotopically grafted along the neuraxis before the onset of migration. This results in cells of the graft following the pathway and colonizing the target tissue and differentiating according to their new position (see Le Douarin, 1982; Le Douarin and Smith, 1988, and references therein). The notion that these undifferentiated cells are multipotent was indicated by the fact that a single neural crest cell was able to generate almost all derivatives, both in vivo and in vitro (Sieber-Blum and Cohen, 1980; Baroffio et al., 1988; Bronner-Frasier and Frasier, 1988; Dupin et al., 1990a; Frank and Sanes, 1991). The study of neural crest differentiation so far suggests a model in which the initially naive, multipotent neural crest cells undergo sequential fate choices and commit themselves to a differentiated cell type in the final location. Although not an obvious trait, the plasticity of the neural crest population remains until at least the second half of gestation. Using heteroexplant methods, the glia of nodose ganglion were marked and subsequent secondary explantation revealed that the glial cells were able to generate cells of neuronal lineage (Ayer-Le Lievre and Le Douarin, 1982; Fontaine-Perus et a l, 1988), After reaching their target site, some of the neural crest derivatives in the skin were still pluripotent (Richardson and Sieber-Blum, 1993; Sieber-Blum et al., 1993). Walter (1994) reported, even in the postnatal sciatic nerve, glial cells were able to generate neurons of various phenotypes, when environmental factors permitted. This plasticity seems to be intrinsic to the cells originating from the dorsal part of the spinal cord (Korade and Frank, 1996). The nature of this plasticity is still not clear. The existence of a multipotent stem cells is generally proposed (Fontain-

50 Perns et a l, 1988; Richardson and Sieber-Blum, 1993; Walter, 1994), and it is supported by the isolation of a progenitor cell which gives rise to both neurons and glia in culture. It was thought to be a stem cell because of its self-renewing ability (Stemple and Anderson, 1992), while alternatively a reversion of putative commited cells to a stem cell phenotype cannot be excluded.

During a subsequent developmental stage, some cells of restricted fate were shown to be already present during the migration of the neural crest and in the earliest stages of DRG neurogenesis. The extent of differentiation during the migration period was documented utilizing various approaches. In back-transplantation experiments the cranial neural crest population is able to generate most types of derivatives according to their enviromental cues, while cells from the trunk area would not give rise to cartilaginous, bone and connective tissue derivatives (see Le Douarin, 1982). Immunolabelling using different monoclonal antibodies suggested that cells of different phenotypes already exist in the migratory population (Girdelstone and Weston, 1985; Vogel and Weston, 1988; Barald, 1988 a, b; Heath et al., 1992). Cell culture experiments suggested that cells with a more restricted developmental potential and cells apparently committed to a particular cell lineage were observed (Sieber-Blum and Cohen, 1980; Ito et al., 1993, Sieber-Blum et al, 1993; Sellect et al, 1993). Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes (Ericksonand Goins, 1995). In fact, an in vitro model of neural crest outgrowth shows that completely distinct neurogenic and melanogenic precursors exist almost immediately after emigration (Henion and Weston, 1997). Segregation of developmentally distinct precursor subpopulations prior to migration therefore might play a role in the appearance of distinct neural crest derivatives in different embryonic locations (Reedy et al, 1997).

1.8.6 The lineage relationship of the neural crest derivatives The diversity of the derivatives of neural crest and the sequential restriction of the cell fate has often been compared to haemopoesis (Metcalf, 1989; Anderson, 1989). In the process of haemopoeisis self-renewing stem cells give rise to a battery of oligopotent precursors, these precursors give rise to unipotent precursors, and cells of defined

51 phenotypes were generated from these precursors (Till and McCullogh, 1961; Suda et al., 1983; Just et al., 1991). Several precursors of different potential in the migrating neural crest have been identified, and the results indicate that some of the cell lineages were more correlated than others. Although the phylogeny of the neural crest cells is still unclear, several lines of observation were quite consistent. Premigratory cells in the dorsal neural tube were able to give rise to both cells of the PNS and CNS, indicating lineage relationship of the neural crest derivatives and some cells in the neural tube (Bronner-Fraser and Fraser, 1988). The second lineage separation takes place when the mesenectomal lineage (i.e. the cartilage and muscle cells of the head) separates from the others. The difference in potential to generate cartilage and muscle cells between the neural crest of head and the trunk is well known (Le Douarin, 1982). There has been some dispute over the ontogeny of cells of the mesenectomal lineage for some time (Baroffio et al., 1988; 1991), but the finding of a single totipotent cell generating cartilage, neuron, glial and melanocytes directly demonstrates the these lineages can derive from a common neural crest precursor cell (Baroffio et al., 1991). As for the fate restricted precursors, research by Le Douarin and colleagues suggests that pluripotent neural crest progenitors give rise to the precursors of neural, melanocytic and mes ectodermal cells by a process involving stochastic restrictions of their developmental potentialities (Baroffio and Blot, 1992; Le Douarin and Dupin, 1993); on the other hand studies by Weston and colleagues hold that the neurogenic and melanogenic sublineages are segregated almost immediately after emergence, while the generation of glial cells is not independent of both lineages (Henion and Weston, 1997). Although the generation of the glial lineage remains undetermined, a consensus emerges from these studies; firstly, the divergence of mesenecetodermal and melanogenic lineages takes place earlier than the separation of the neuronal and glial lineages (Baroffio and Blot, 1992; Erickson and Gois, 1995). Secondly, a precursor cell for neurons, glia and melanocytes exists, although very rare (Baroffio et al., 1988; Dupin et al., 1990a; Bronner-Fraser and Fraser, 1988; Henion and Weston, 1997). Thirdly, the glial lineage is the last to be separated from any kind of bipotential precursors, including sensory/glial, adrenergic/glial, melano/glial, cartilage/glial precursors (Dupin et al., 1990a; Le Douarin and Dupin, 1993; Henion and Weston, 1997).

52 Within the neuronal lineage several types of sublineage were reported. In the head area the neurons orignate from two sources, the placode and the neural crest (Narayanan and Narayanan, 1980; D'Amico-Martel and Noden, 1983; Le Douarin et al., 1986; Noden, 1993) The main criteria for detecting neural crest derived neurons includes the sensory neurons, which could be detected by VIP (vasoactive intestinal peptide) or SP (substance P) (for details see Dupin et al. 1990b), and cells of the sympathoadrenal lineage, which includes sympathetic neurons, SIF cells and chromaffin cells of adrenal medulla (for review see Anderson 1993), which can be detected by antibody against TH (tyrosinehydroxylase) (Anderson et al, 1991). Recently a new sympathoenteric lineage was described. These cells depend on c-ret for survival while sympathoadrenergic cells do not (Durbec et al, 1996). The generation of subtypes of neurons from a single cell seems to be non-stochastic. For example, the generaton of adrenergic cells and sensory neurons were correlated when tested statistically (Baroffio and Blot, 1992). This was also confirmed by the observation that in a clonal analysis no homogenous clones of adrenergic cells, which were supposedly derived from a commited adrenergic precursor, were ever found (Le Douarin and Dupin, 1993). As for sub-types of PNS glial cells, Dupin et al (1990a) propose that there are distinct precursors for Schwann cells and satellite cells of DRG. Rüdel and Rohrer (1992) showed that sensory and sympathetic satellite cells express different cell surface antigens.

1.9 Gliogenesis and neurogenesis during DRG development Lineage analysis studies have shown that the migrating neural crest cells are a mixture of committed progenitors and multipotent precursors (Baroffio et al., 1991; Bronner-Fraser and Fraser, 1988; Siber-Blum, 1989a; Dupin et al, 1990a). From retroviral lineage tracing and clonal analyses of cells from symapathetic and sensory ganglia it was found that during gangliogenesis neuronal and glia cell precursors diverge shortly after gangliogenesis, yet prior to their last division (Duff et al., 1991; Hall and Landis, 1991, 1992). However new evidence shows that distinct fate- restricted neuronal and glial precursors from neuron-glia progenitors occcur after one day of emigration of the crest from the neural tube (Henion and Weston, 1997). One

53 way or the other, the cells of aggregating ganglia are supposedly in a more advanced stage than the migrating neural crest, which is reflected by the huge predominance of committed progenitors of neuron and glia, and a very small number of multipotent precursors in the nascent ganglion (Fontaine-Perus et al., 1988; Hall and Landis, 1991, 1992). Neuronal and glial fates segregate well before overt neuronal differentiation, and fate restricted neuronal precursors are still mitotically active (Henion and Weston, 1997; Memberg and Hall, 1994). Some neuronal proteins are expressed soon after the decision of neuronal fate. These include an epitope recognized by SNl antibody, Hu neuronal protein, neuroD and neurogenin (Marusich et al., 1986, 1994; Lee et al., 1995; Ma et al., 1996). During gangliogenesis and before final division, the DRG neurons are bipolar and start to express the earliest mature phenotypic traits, including catecholamine synthetic enzymes (only in the SCO) (Hall and Landis, 1992), p-tubulin type 3 and low and intermediate molecular weight neurofilament proteins (Memberg and Hall, 1994; Chen and Chiu, 1992). Other molecules that are expressed in the proliferating and early post-mitotic peripheral neurons include epitopes recognized by B30/33, Brn-3.0 ,c-ret, MASH-1,

Nkx-5.1, P 2X3 gated channel, SSEA-1, polysialytated NCAM (PSANCAM) (Stainier and Gilbert, 1990; Stainier et al., 1991; Fedtsova and Turner, 1995; Pachnis et al., 1993; Lo and Anderson, 1995; Durbec et al., 1996; Lo et al., 1994, Bober et al., 1994; C. C. Chen and J. Wood, personal communication; Sieber-Blum, 1989b; Boisseau et al., 1991). The neurons in the rat embryonic SCG have their final division at E l4, while the peak of division for the glioblasts is between E l6-18, which is after the peak of neurogensis (Hall and Landis, 1991; 1992). At this stage the sensory neurons are still capable of proliferation while ciliary neurons stop proliferation and start to differentiate at this stage (Rothman et al., 1978; Rohrer and Thoenen, 1987).

Very few glial phenotypes have been identified at this stage. This lack of markers is not due to the uncommitted nature of glia, as the choice between the neuronal and glial fates was complete in most of the cells before the formation of the ganglion (Hall and Landis, 1991). An early but less defined marker for the Schwann cell lineage in quail is the carbohydrate epitope 4B3 (Cameron-Curry et al., 1991). It is expressed in both the CNS and PNS glia, and its reactivity is associated with a

54 carbohydrate determinant shared by several glia surface antigens including a subset of SMP molecules (Cameron-Curry et al, 1991; see below). It is expressed in the migrating neural crest in E2.5 quail (Cameron-Curry et al, 1993). Whether this carbohydrate epitope is conserved in rat is not documented. SMP (Schwann Cell Myelin Protein) is a molecule expressed in the external membranes of quail Schwann cells which shares 80 % homology to the mouse MAG myelin protein in the transmembrane domains (Dulac et al, 1988; Cameron-Curry et a l, 1991; Dulac et al, 1992). The immature satellite cells in the aggregating ganglion express the 4B3+ epitope, which is carried from early developmental stages onward by all glial cells of the PNS (Cameron-Curry et al, 1991). SMP is an early marker for the Schwann cells in the quail system but is absent from the mature satellite cells (Dulac et al, 1988). When cultured in vitro with no added growth factor, the satellite cells express SMP rapidly, demonstrating that SMP expression is a PNS glia marker but is more susceptible to reversible regulation from the environment (Cameron-Curry et al, 1993). It is known that glial cell differentiation is modulated by interactions with neuronal elements (Pannese et al, 1972; Aguayo et al, 1976; Bunge et al., 1980; Salzer et al, 1980; Bray et al, 1981; Lemke and Chao, 1988; lessen et al., 1990; Rudel and Rohrer, 1992). Interestingly the neuronal soma and axons regulate SMP expression in the quail glial cells in opposite directions. Close contact with the neuronal soma results in the down-regulation of this otherwise constitutively expressed phenotype, and contact with neurites results in up-regulation (Cameron- Curry et al., 1993; Pruginin-Bluger et al., 1997).

1.10 Axon/Schwann cell interactions during the formation of nerves The relationship between axons and Schwann cells during development of peripheral nerves in the rat was studied by several groups using different nerves as their models (Harrison, 1924; Peters and Muir, 1958; Ziskind-Conhaim, 1988; Carpenter and Hollyday, 1992a, b; lessen et al. 1994). Generally the larger the axon diameter is, and the more proximal to the spinal cord the nerve is, the more advanced development is. There were distinctions made between motor and sensory nerves in these studies. Some authors have proposed role of guiding peripheral neurite outgrowth for embryonic Schwann cells, based on the timing of their presence in the nerves (Noakes

55 and Bennett, 1987). However, in both mice which lack neuregulin or its receptor ErbB3 (see below), cells of Schwann cell lineage are almost absent in the nerves from the precursor stage, but the targeting of the axons appears to be normal (Meyer and Birchmeier, 1995; Riethmacher et al., 1997). The lack of Schwann cells in the Splotch mouse, which has mutations in the Pax-3 gene and defects in neural crest derivatives (Epstein et al., 1991; Franz and Kothary, 1993), does not result in failure of motor outgrowth. These experiments reinforce the hypothesis that the nerves do not need Schwann cells as guide posts to targeting the innervation field (Grim et al., 1992), like the radial glial in the CNS. Rather, the presence of neurite outgrowth from the ventral motor neuron seems to be essential for attracting Schwann cells to their final position in the nerves (Bhattacharyya et al., 1994).

1.11 The Schwann cell precursor At day 13 to 14 of gestation the sciatic nerves of rat start to innervate the hindlimb (Reynold et al., 1991). The nerves are composed of tightly packed, unmyelinated axons and growth cones. Schwann cell nuclei are both at the periphery and within the nerve, while the cytoplasm of these Schwann cells wraps around the axons and forms a continuous barrier from the surrounding mesenchymal connective tissue around the nerve (lessen et al., 1994). Growth cones disappear at around E l 6 and hence there is a reduction in the nerve diameter (Ziskind-Conhaim, 1988). The Schwann cells extend digits between the packed axons and gradually separate them into groups from E14 onwards (lessen et al., 1994). There is a massive increase of Schwann cell division rate around E l8-19 though cell division in precursors is substantial (Stewart et al, 1993). As a result the ratio of Schwann cells and axons falls gradually and the establishment of a 1:1 relationship of Schwann cells and axons of larger diameter, starting from the periphery of the nerve, is seen shortly before birth (Ziskind- Conhaim, 1988; Peters and Muir, 1958). The glial cells associated with the axon projection in the rat hind limb between El3 and 14 are morphologically and phenotypically distinct from their forerunners, neural crest cells and from cells dissociated from the sciatic nerves of E16-17 in several ways. They have a flattened morphology in vitro, higher motility rate than older Schwann cells, but already express most of the proteins that characterise non-myelinating Schwann cells in adult

56 nerves (lessen et al., 1994). As mentioned earlier, phenotypically, although they are p75NGF-R positive, they do not express SI00 as do cells from nerves that are 2 days older. They undergo programmed death in culture within 20 hours in conditions which support older embryonic Schwann cells, but can be rescued by axonally derived neuregulins (Gavrilovic et al., 1995; lessen et al., 1994). Furthermore, they are different from neural crest in that under certain culture conditions the crest cells lack GAP-43 expression, which is present under the same conditions in the Schwann cell precursor (lessen et al., 1994). Mouse peripheral nerves also contain Schwann cell precursors, but they are present at E l2 and E l3, two days earlier than in the rat, and the precursor to Schwann cell transition occurs in the developing sciatic nerve between E14 to E15 (Dong, Sinanan, Mirsky, lessen, unpublished).

1.11.1 The Schwann cell precursor is a major regulator of nerve development during early development of PNS A striking hypothesis has been proposed that the survival of major neuronal populations, including DRG neurones and motor neurones, depends on trophic support from Schwann cell precursors and early Schwann cells (Riethmacher et al, 1997; lessen and Mirsky, 1998). Though it is long established that the sensory neurons are independent of growth factors before the axons reach their target, it is now known that in vitro sensory neurons require growth factor support prior to target innervation (Henderson, 1996; Davies, 1997), most probably provided by the Schwann cell precursor. Strong evidence for the early trophic role of Schwann cell precursors comes from the observation that the ErbB3 (see following) knock-out mice, which lack Schwann cell precursors and Schwann cells, also lost most of their sensory and motor neurons during the second half of embryonic development. DRG neurones were reduced by 80% in number between E l2 and E l4 and motor neurones between E l6 and 18 (Reithmacher et al., 1997). The death of the DRG neurones is unrelated to their target dependency, since the ErbB-3 related death takes place before most sensory axons reach their target field (Davies, 1994; Lewin and Barde, 1996). Experiments on chimeric animals also argue against the possibility that the DRG or motor neurones died because of lack of ErbB3 receptors on the neurones themselves.

57 suggesting that the loss of DRG neurones is due to the loss of Schwann cell precursors rather than ErbB3 mediated signalling.

1.12 The CNS/PNS transitional zone The CNS-PNS transitional zone (TZ) is one of the primary partitions of the nervous system, and is present at the attachment of all nerves to the CNS except for the olfactory and vomeronasal nerves. It separates the CNS from the PNS environments and marks a sharp discontinuity of tissue types (Fraher, 1992; Doucette, 1991). On the CNS side of this zone the myelinated fibers are separated by astrocytes and a barrier is formed primarily of interdigitating astrocyte processes. The astrocyte glia limitans extends across the axon bundle comprising the rootlet (Golding and Cohen, 1997). The glia interface arises at the interface of the neural tube, probably by interaction between cells derived from neural crest, the processes of neuroepithelial cells, and the basal lamina of the neural tube, (reviewed by Golding et al., 1997; Fraher, 1997). The organisation of motor exit zone and sensory innervation entry zone, the two main types of CNS-PNS TZ, differ in the head and trunk region. Firstly in the trunk area the motor axons and sensory axons exit/enter at different dorsal- ventral levels of the neuroepithelium, while in the hindbrain the sensory and motor element of the cranial nerves utilizes the same exit/entry point (reviewed by Golding et al., 1997). A further difference in arrangement lies in the motor axons. Axons of spinal motor neurons grow away from the floor plate and exit the neural tube in a continuous band along its anterioposterior axis at a specific dorsoventral level. Once in the periphery, motor axons form nerve bundles due to instructions from the adjacent segmented mesoderm and follow a course only through the anterior half of the somite (Keynes and Stern, 1984). In the hindbrain motor axons of the cranial nerves grow anteriorly and dorsally and remain in the neural tube for some distance before exit into the periphery through defined points in even numbered rhombomeres (Lumsden and Keynes, 1989). Study before the outgrowth of motor axons shows that late emigrating neural crest cells migrate to the exit point of cranial branchiomotor nerves and form boundary cap cells (Niederlander and Lumsden, 1996). At this stage the exit zone can be distinguished by its c-cad7 mRNA expression, a calcium- dependent cell adhesion molecule (Nakagawa and Takeichi, 1995; Niederlander and

58 Lumsden, 1996), although the protein is detected in premigratory and a subset of migrating neural crest (Nakagawa and Takeichi, 1998). Late surviving boundary cap cells were suggested to regulate sensory afferent ingrowth (Golding and Cohen, 1997). Cell surface molecules in the neuroepithelium do not seem to be required for motor axon guidance in that outgrowth of motor axons remain unchanged when the odd numbered rhombomere is inverted in anterio-posterior axis (Guthrie and Lumsden, 1992). The entry/exit zone is determined by a stepwise guidance, probably involving a segmentation pattern within the neuroepithelium and attractant/repellent factors in the periphery. The area where the primary sensory neurones enter and innervate the CNS is known as the dorsal root entry zone (DREZ) (for review see Golding et al., 1997). The glial cells residing in the DREZ, the boundary cap cells, develop expression of Krox-20 and SlOO precociously, ahead of the PNS glia (Murphy et al., 1996). This region has attracted special interest in an attempt to understand the factors contributing to the regeneration and re-entry of sensory axons after injury. After dorsal root crush adult axons will regenerate only within the dorsal root as far as the segment covered by Schwann cells (Cajal, 1928; Carlstedt, 1985). On the contrary, regenerating axons are able to enter the spinal cord in neonatal rats up to 1 week old (Carlstedt et al., 1987; Carlstedt, 1988). A study using an in vitro model of the rat DREZ shows that the ability of neurites to cross the DREZ depends on both properties related to the age of the sensory neurons and the spinal cord (Golding et al., 1996).

1.13 The gut The enteric nervous system (ENS) is a part of the autonomic nervous system. Its major component is distributed in the wall of gastrointestinal tract. There are two major plexuses, the myenteric (or Auerbach’s) plexus which located between the circular and logitudinal muscle layers, and submucous (or Meissner’s) plexus, which is in the submucosa. The ganglia consist of neuronal cell bodies and enteric glia, with interconnected nerve bundles containing axons and enteric glia. The extrinsic neuronal projections from sensory and autonomic ganglia to the bowel are few but important in regulation of gastric motility (Gab el la, 1979). In terms of its morphology, physiology, and neurochemical characteristics, the ENS is quite

59 different from other elements of the PNS and more closely resembles the CNS. Unlike the rest of the PNS the enteric plexuses contain no large connective tissue spaces or blood vessels and are not enclosed in perineurial sheaths (Gabella, 1972). Most of the neurones of the ENS are not directly innervated by a preganglionic input from the brain or spinal cord, and the ENS can mediate reflex activity in the complete absence of CNS input (Gershon, 1981; Gershon and Erde, 1981). Each of the classes of neurotransmitter found in the brain are represented in the ENS (Furness et al., 1992; Gershon, 1990). The ENS arises from the rhombencephalic (vagal) neural crest, which is derived from the neural tube between somites 3-6 (originally thought to be 1-7) and from the sacral crest located posterior to somite 28 (Yntema and Hammond, 1954; LeDouarin and Teillet, 1973; Serbedzija et al., 1991; Peters-van der Sanden et al., 1993; Epstein et al., 1994). Recently an additional source has been identified to originate from the truncal crest and colonize only the rostral forgeut. The neuroblasts from this region, unlike the other two, are ret-GDNF-independent (Durbec et al., 1996). The migration of the neural crest into the gut has been studied using various tracer techniques (Serbedzija et al., 1991; Peters-van der Sanden et al., 1993; Epstein et al., 1994) and molecular markers including neurofilament, NC-1 and E/C8-1, GFAP, HNK-1, laminin binding protein, dopamine-p-hydroxylase (BDH), and ANNA-1 (Hu) (Payette et al., 1984; Tucker et al., 1986; Rothman et al., 1986; Epstein et al., 1991; Pomeranz et al., 1991; Coventry et al., 1994; F airman et al., 1995) (for a review see Gershon, 1997).

Some factors that are crucial during the development of the ENS have been identified by studying natural and targeted mutations. One of the earliest-acting of these is the glial cell line derived neurotrophic factor (GDNF) and its receptor. Ret (Trupp et al., 1996; Treanor et al., 1996). Ret is encoded by the c-ret proto-oncogene, which is transiently expressed by enteric neural precursors (Lo and Anderson, 1995; Pachnis et al, 1993).When c-ret is knocked out, the bowel contains no neurons or glia except for those in the esophagus and upper stomach (Durbec et al., 1996; Shuchardt et al., 1994). Similar symptoms are seen in GDNF knock out mice (Moore et al., 1996; Pichel et al., 1996; Senchez et al., 1996; Durbec et al., 1996). Mash-1, a basic helix- loop-helix transcription factor, is the mammlian homologue of achaete-scute in

60 Drosophila (Lo et al., 1994; Guillemot and Joyner, 1993). It is expressed by enteric neural precursors, sympathoadrenal cells, and thyroid parafollicular cells (Lo et al., 1994; Guillemot and Joyner, 1993; Clark et al., 1995). Targeted mutations of Mash-1 result in the near-total loss of sympathetic neurons and the complete loss of the early- developing subset of enteric neurons (Guillemot et al., 1993; Blaugrund et al., 1996). Based on the ret-GDNF dependency, and the close lineage relationship of the enteric neuron and sympathetic neurons (Anderson, 1993), two lineages have been proposed for the development of ENS. The sympathoenteric lineage gives rise to a majority of the ENS and the superior cervical ganglion, while the sympathoadrenal lineage forms the caudal sympathetic nervous system, the adrenal medulla, and the ENS of the rostral foregut (Durbec et al., 1996; Balugrund et al., 1996). Other factors which are important in the development of ENS include neurotrophin 3 (NT-3) (Farias et al., 1994), ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF) (Gershon, 1997) and endothelin 3 (EDN-3) (Baynash et al., 1994; Rothman and Gershon, 1984; Kapur et al., 1993). The glial cells in the ENS share many similarities with the other types of PNS glia. However, there are several differences between the ENS glial cells and Schwann cells. The enteric glia cells resemble CNS glia cells in that they do not secrete an individual basal lamina, have a starlike multiprocessed outline and contain high levels of glial fibrillary acidic protein similar to that found in astrocytes (Cook and Burnstock, 1976; Jessen and Mirsky, 1980). Some molecules expressed by the Schwann cells, such as p75 NGF-R, Gal-C and SMP, are not detected in enteric glia in vivo (Bannerman et al., 1986, 1988; Jessen and Mirsky, 1983; Le Douarin and Ziller, 1993). However, these Schwann cell phenotypes are shown to be expressed in embryonic precursors and when cultured in vitro without enteric influence, showing a reversible regulatory signal for the phenotypes in the enteric environment (Jessen and Mirsky, 1983; Cameron-Curry et al., 1993; Bothwell, 1996). Furthermore, unlike Schwann cells, some enteric glia express cell surface gagliosides recognized by the antibodies A2B5 and LBl (Bannerman et al., 1988). Other differences include the fact that the enteric glia have a higher basal level of proliferation than Schwann cells under the same conditions (Eccleston et al.,

1987a).

61 1.14 Placode 1.14.1 Origin The placodes are discrete regions of thickened columnar epithelium on the head that are induced to form by different regions of the neural tube (Jacobson and Sater, 1988). They appear anteriorly and laterally with respect to the neural plate and neural folds, which give rise to CNS and PNS respectively. More caudally the superficial cephalic epithelium also generates the neurogenic placodes from which epithelial cells detach and migrate to give rise to certain canial ganglia. The olfactory, adenohypophyseal and trigeminal placodes originate within the neural folds (Couly and Le Douarin, 1985, 1987, 1990; Eagleson and Harris, 1990), but some researchers hold that they could well originate within the epidermis (Stark et al., 1997). The otic, lateral line, and epibranchial placodes clearly form from the ectoderm adjacent to the neural tube.

1.14.2 Organogenesis These thickenings of the body wall are transient structures which exist during the organogenesis of the embryo and give rise to various sensory organs. The lens placode originates from the CNS and contributes to the eye. The olfactory placode develops into olfactory epithelium, where the receptor neurons for the sense of smell reside. The olfactory epithelium also gives rise to olfactory glia, the only glial cells derived from the placode. The otic placode invaginates and forms the otic vesicle, from which the inner ear and the 7th and 8th ganglion are derived exclusively. The lateral line placode gives rise to the fins in fish and is not present in higher vertebrates. The trigeminal placode contributes to the primary sensory neurons in the trigeminal ganglion. Epibranchial placodes are located near the pharyngeal pouches and they give rise to petrosal (9th) and nodose (10th) ganglia. Some placodes are only present in certain species. For example, the five lateral line placodes (two pre-otic and three post-otic) in the fish give rise to neuroblasts that form a lateral line cranial nerve, which subsequently innervates receptors of the lateral line system that also arise from these placodes (Northcutt et al., 1994).

1.14.3 Contribution to cranial ganglia

62 Placode derivatives include ciliated sensory receptors, sensory neurons, neuroendocrine and endocrine cells, glial and other suporting cells (table 1-2) (reviewed by Baker and Bronner-Fraser, 1997). Neurogenesis has been studied more than other derivatives. Sensory neurons differentiate from progenitor cells that originate from a thickening of the embryonic ectoderm, i.e., the neural crest and neurogenic placodes. In avia and mammals the neurons of DRG and certain cranial sensory ganglia orginate from the neural crest, whereas the neurons of the remaining cranial sensory ganglia originate from neurogenic placodes. The trigeminal placode and epibranchial placodes give rise to the cranial ganglia. They do so by delamination from the placode and migrate to their final destination, a process in some way rather similar to their neural crest derived counterparts (Noden, 1978; Narayanan and Narayanan, 1980; D ’Amico-Martel and Noden, 1983; Le Douarin et al., 1986). Placode-derived neurons start differentiating before the neural crest-derived neurons and are initially larger than these neurons. They usually occupy more central locations in the ganglion than the neural crest derived neurons, presumably due to the early onset of migration, and the difference in the morphology remains in adulthood (Noden, 1978; D’Amico-Martel and Noden, 1983). Table 1-3 shows the contribution of placode and neural crest to the cranial ganglia (adopted from Le Douarin et al., 1986).

Table 1-2 Derivatives of neural crest and placodes, cateaorized by cell type

Derivatives Placode Neural crest Ciliary receptors + Olfactory, otic, lateral line (+) Some lateral line Neurons Sensory + +

Autonomic - +

Enteric - + Glia + Olfactory (Schwann) + Neuroendocrine + Olfactory (GnRH) adenohypophysis Chromaffin (adrenalin), C-cells /endocrine (pituitary) (calcitonin) ECM-secretmg +lateral line (cupula) +Dermis, cartilage, bone, dentine

Smooth muscle - + Melanocytes - +

GnRH, gonadotropin-releasing hormone. Source; Baker and Bronner-Fraser, 1997

63 Table 1-3 contribution of placode and neural crest to the cranial ganglia

Neural crest-derived Placode-derived dorsomediai part of the trigeminal ganglion ventrolateral part of the trigeminal ganglion trigeminal mesencephalic nucleus (CNS) geniculate ganglion root ganglion of facial nerve (*) vestibular ganglion root ganglion of the glossopharyngeal nerve acoustic ganglion jugular ganglion petrosal ganglion dorsal root ganglia nodose ganglion

* embedded in the vestibular ganglion Source: Le Douarin et al., 1986

1.14.4 Comparison of the placode and the neural crest Placodes seem to be induced by particular regions of the neural tube and are confined to the head, except the most anterior olfactory and adenohypophyseal placodes, while neural crest cells form at the interface of neural plate and epidermis along the whole rostro-caudal axis (Baker and Bronner-Fraser, 1997). The cell types derived from these two structures differ as well. Placodes do not normally form melanocytes or autonomic neurons, or produce mineralized matrices like bone and dentine, and neural crest very seldom forms ciliated sensory receptors (Baker and Bronner-Fraser, 1997). Their neuronal derivatives differ morphologically. The placode-derived neurons are large, and deeply silver-impregnated, and are not densely packed. The nuclei are spherical, eccentric and are ususally in contact with the wall of the perikaryon at one side of the cell. The neural crest-derived ones are smaller and stain more lightly, and the population density is considerably higher. Their nuclei are spherical or slightly elliptical. The placode-derived cranial sensory neuons of the vestibular and nodose ganglia in avian embryos exhibit differences in neurite growth rate and the duration of neurotrophin-independent survival in vitro that arise prior to gangliongenesis and target contact (Davies, 1989; Vogel and Davies, 1991)

1.14.5 Molecular markers to date Torres and Giraldez proposed a model in which specific combinations of genes rather than single gene expression appear to be characteristic for each placode (1998). They suggested that transcription factors of the six. Pax and dix families might contribute to the combinatorial code (see table 1-4). As attention to the molecular phenotypes of placode is recent, comparatively fewer molecules have been identified as placode

64 markers. Of them the Pax gene family has been most studied. Pax-3 expression is suggested to be a good marker for the trigeminal placode (Stark et al., 1997). This expression is shared by the neural crest (Franz and Kothary, 1993). The otic placode expresses the Pax-2 gene (Nornes et al., 1990; Rinkwitz-Brandt et al., 1996). Delta-1, which is involved in neurogenesis, is considered to be an early marker for placode- derived neurons in the cranial ganglia (Anthony Graham, personal communication). Cytokeratin, AEl and CAMS.2 have been recently characterized as markers specific for embryonic sensory neurons which are derived from neurogenic placodes (Okabe et al., 1997). However this immunoreactivity disappeared during the late embryonic to post-embryonic stage and was not observed in the roots of these ganglia in the postnatal stage. No other peripheral neurons have cytokeratin during development, nor does the neural crest.

Table 1-4 Transcription factor expression in sensory placodes

six3 Pax6 dlx3 sixl Lens + + Ear + + Nose + + + +

Source; Torres and Giraldez, 1998

1.15 The ear l.lS.lGeneral anatomy and corresponding functions of the inner ear The ear consists of three major parts, which include the external part of the auricle and meatus, the middle part or tympanic cavity, and the internal part of the vestibule, cochlea and semicircular canals. The inner ear is the sensory organ responsible for the senses of hearing, balance and detection of acceleration in vertebrates. The inner ear of higher vertebrates contains six to eight sensory organs of three different types; the cristae, for detecting angular acceleration (found in the semicircular canals); the maculae, for detecting linear acceleration or gravity (found in the saccule, utricle and lagena); and an organ for detecting sound (called the organ of Corti in mammals and the basilar papilla in non-mammals) (see figure 1-4). These mini sensory organs

65 consist of highly topological specific arrangements of heterogenic cells, and are sometimes referred to collectively as the sensory epithelium. They collaborate with the fluid filling the inside of the bony outer structure, to carry out the function of the inner ear. Stimuli related to vestibular functions are produced by movement of the endolymph through the semicircular canals and by the position of the otoliths within the utricle and saccule, which detect the direction and speed of the circulating fluid and otolith movements. Sound waves are transmitted to the inner ear through the middle ear and converted to mechanical vibrations of the endolymphatic fluid, which results in the stimulation of the auditory hair cells and the generation of electrical activity (see Dallos 1992 for mechanism of hearing). The hair cells are innervated by sensory neurons that project toward the vestibular and auditory nuclei in the brainstem. The cell bodies of these nerves lies in the 7th and 8th (vestibular and acoustic) ganglia.

1.15.2 The structure of sensory organs and their cell types The cristae are located at the enlarged end of the semicircular canals (the ampulla). They consist of receptor hair cells, supported by neighboring nonsensory cells. The hairlike processes of the receptor cells are embedded in a sugar protein mass, the cupola. The maculae are located within the utricle and saccule walls. They consist of a long row of hair ells and supporting elements. The hair-like processes are embedded in a gelatinous (protein-sugar) layer which contains calcium salts (otoliths). Two types of hair cells are present in the vestibular organ; type 1 hair cells that are pear shaped and are innervated by afferent nerve endings, and type 2 hair cells that are cylindrical and innervated by afferent and efferent fibres (see for example, Fawcett, 1986). The type 1 cells are predominatly located in the central region of the sensory epithelium while type 2 are at the periphery. The organ of Corti consists of hair cells and support cells, with pillar cells in the middle dividing them into an outer and inner population (figSB). The ‘tall’ inner hair cells (IHC) have exclusively afferent innervation, while the ‘short’ outer hair cells (OHC) are also innervated by efferent nerves (for a comprehensive review for hair cell types see Forge et al., 1997). During sound stimulation the sensory hair cells are stimulated by a movement between the top surface of the organ of Corti and the tectorial membrane. The efferent innervated

66 hair cells probably modulate the signal that reaches the afferent innervated receptor cells. The organ of Corti of mammals is located in the middle compartment of the cochlear duct, the seal a media (see figure 5 A) (Steel and Brown, 1994). The membrane separating the scala media and the scala vestibuli is called the Reissner’s membrane. The stria vascularis on the lateral wall of the cochlear duct, and probably the Reissner’s membrane (fig5A,B), has a key role in generating the high resting endocochlear potential in the endolymph that fills the scala media, and it also controls the high and low Na^ concentration in the endolymph (Dallos, 1992; Fermin, 1995; Steel and Brown, 1994). It should be noted that the structure of the chick and rat cochleas are different. In chick the sensory hair cells for hearing are located in the basilar papilla. Opposite the basilar papilla of chick on the wall of the cochlea duct there is the tegmentum vasculosum (TV). In the chick the TV is believed to be responsible for maintaining the ionic difference between the endo- and epi-lymph, the same function as Reissner’s membrane and the stria vascularis in the rat (Bissonnette and Fekete, 1996). The endolymphatic duct and sac channel the fluid out of the ear to the ventricular system of the brain. The semicircular canals and the rest of the epithelium are lined with simple cuboidal epithelial cells.

1.15.3 The generation of the inner ear Generation of the inner ear involves a wide range of cellular phenotypes whose formation implies a series of cel I-fate decisions during otocyst differentiation. In addition, several phenotypes are segregated into specialized areas in a highly organized fashion. What factors and how they contribute to the complicated formation is still largely unknown. During the past few years a large number of genes expressed during the early stages of inner ear development have been identified (for review see Fekete 1996; Torres and Giraldez, 1998). The expression of these genes has been speculated to be associated with the morphogenesis of structures such as the semicircular canals (e.g. Nkx-5.1), or sensory organs (bone morphogenic protein 4, BMP-4) (see below). The collective expression of some of these markers were suggested to map the otic vesicle and provide reference to the morphogenesis or designation of cell types (Feteke, 1996, see following). Although the proposed explanation still lacks sound support, it is now generally agreed that heterogeneity

67 already exists before overt differentiation occurs during the otic pit and otic vesicle stage, be it of axis determination or cell specification. The following section will discuss the known facts about the gross morphological changes, regulation of otic induction, and the mechanisms of regional specification, followed by a discussion of how different cell types differentiate in the inner ear.

1.15.3.1 Early morphological changes The first identifiable stage of inner ear development begins with the formation of the otic placode from the surface ectoderm of the embryo. It can already be recognized in apposition to the neural tube in the 3-6 somite embryo (reviewed by Torres and Giraldez, 1998). The otic placode then invaginates to form the otic cup and then the otic vesicle (Anniko, 1983; reviewed by Feteke, 1996; Torres and Giraldez, 1998). Neuroblasts have been seen delaminating from the otic cup of the zebrafish embryo to form the cochleovestibular ganglion (Haddon and Lewis, 1996). It is not directly proved but the generation of cochlear and vestibular ganglion in mammals is believed to be similar (Torres and Geraldez, 1998). As development proceeds this single ganglion then splits into the cochlear ganglion and vestibular ganglion (reviewed by Torres and Geraldez). The endolympatic duct is the first among the structures to elongate from the oval shaped vesicle as it matures, E4 (stage22) in the chick (Bissonnette and Fekete, 1996) and El2 of rat (Wu and Oh, 1996; MJ Lee, unpublished). This is followed by the elongation of the cochlear duct at E5 (stage24) of chick (Lillie, 1952; Bissonnette and Fekete, 1996), and E l3 of rat (M-J Lee, unpublished). The pouch-like anlagen of the semicircular canals develops into its final tubular morphology very quickly during E6 of chick (Bissonnette and Fekete, 1996). The specialization of distinct structures is believed to happen before distinct morphological structures can be identified (see below). Morphologically distinct hair cells and supporting cells do not appear until E l7-18 in rat, or E l3-14 of mouse (Ryan, 1997).

1.15.3.2 Regional specification The determination of the polarity of the otic vesicle takes place early, soon after the induction of the otic placode. Different CAMs, which are believed to expressed in

68 specific modal patterns, are differentially expressed in otic placode as early as the beginning of the induction (Richardson et al, 1987). Based on the pattern of gene expression of several pattern formation related genes, Fekete proposed a boundary theory for regional specification (see above). The model states that at the otic pit and/or otic vesicle stage, the vesicle is subdivided by boundaries into compartments. The boundaries are established at the limits of gene expression domains, or by other secreted inductive factors from adjacent tissue, i.e. the neural tube, the notochord, neighbouring ectodermal tissue (Feteke, 1996; Lawrence and Struhl, 1996). The nature of the signal proposed for the latter probably belongs to the same family of secreted factors which determine the dorsal-ventral axis of neural tube, the antennapedia class homeobox genes or other transcription factors that are known to regulate the segmentation of the hindbrain. Three boundaries (mediolateral, anterioposterior, and dorsoventral) and six points of intersection can be identified this way. Sensory-competent cells will become specified to form sensory organs only at the point of intersection or adjacent to boundaries. This supposedly not only specifies the location of the organ but also the type of the organ (Fekete, 1996).

Before distinct morphological structures are observed, expression of some early genes separates into patches and later in development corresponds well to specific sensory structures. Based on this type of observation several genes expressed during early otic development were suggested to be markers for the primordium of certain structures. BMP-4 is suggested to be a marker for the sensory organs, based on the good association of its expression in the gradually recognizable sensory primordium in the chick. p75NGF-R and BDNF are expressed in different patterns on cristae and maculae and can be used to distinguish these two structures (Wu and Oh, 1996). The BMP-4 gene is expressed in the rat inner ear as well (chaper 5). Nkx-5.1 is suggested to be a marker for the semicircular canals in the mouse. It is expressed in the otic vesicle in a restricted dorsal-lateral pattern, and the expression persists in the vestibular structures throughout inner ear development. Mice with a null mutation of Nkx-5.1 fail to develop semicircular canals (Rinkwitz-Brandt et al., 1996; Hadrys et al., 1998). Pax-2 is suggested to be marker for the cochlear duct in the mouse. Apart from these murine genes, in the chick GH-5 and SOHo-1 are suggested to be markers

69 of semi-circular canals, including both sensory and non-sensory parts of the canals (Fekete, 1996).In zebrafish mshC, a member of the homeobox gene family, is expressed only in the maculae (Ekker et al., 1992).

1.15.3.3 The specification of sensory organs versus specification of other structures Interestingly, the specification of polarity of the gross anatomy and the specification of the sensory organ seems to be regulated by different mechanisms, as suggested by the fact that in some mutations when there is severe deformation of the inner ear morphology, sensory organs with distinct hair cells and supporting cells are still present, as in the case when otic epithelium (without surrounding mesenchyme) is transplanted to a non-otic field (Swanson et al., 1990) , or in mice lacking Hoxa-1 or FGF-3, and in kreisler mutant mice (Mark et al., 1993; Chisaka et al., 1992; Mansour et al., 1993; McKay et al., 1996). Early expression of cristae markers suggests that their specification proceeds independently of the canals in which they normally reside. This idea is further supported by the observation that cristae markers are evident even in the abnormal or failed semicircular canal formation in zebrafish mutants eselsohr and znikam (Whitfield et al.,1996; Malicki et al., 1996). In experiments when the otic vesicle in the chick was rotated 180° around its vertical axis in situ, the vesicle expressed Pax-2 in a pattern corresponding to the new surrounding tissue, suggesting that the Pax-2 expression is not determined at the stage of vesicle formation (Linberg et al., 1995).

Recent experiments suggest that the specification of sensory epithelium might occur earlier than the specification of morphological structures. In transplant experiments of otocyst of E2.5 chick, it was shown that whereas the anterior-posterior (A/P) axis of the sensory organs is fixed at the time of transplantation, the A/P axis for most non­ sensory structures, is fixed later (Wu et al., 1998). Different compartments of the inner ear seem to be regulated independently during morphogenesis. This could be demonstrated in the Pax-2 and Nkx-5.1 null mutations. As mentioned above, the Pax-

2 gene is related to the development of cochlear duct while Nkx-5.1 is important in the forming of semicircular canals. Up to mouse ElO the gene expression of Pax-2 is

70 not affected in the Nkx-5.1 knock out mice, nor vice versa, suggesting that the gene which is responsible for defects in forming the semicircular canals is not involved in the formation of the cochlear duct, and vice versa (Hadrys et al., 1998; Torres and Giraldez, 1988). However, a clever mechanism works to impose the framework of morphogenesis on the future sensory area. BMP-4 is expressed at the otic cup stage in two restricted patches beside the neurogenic area. During later development some sensory areas originate by segregation from an initially continuous future sensory area, while others appear de novo, judging from the BMP-4 expression. The fact that cristae and maculae of different structures could be derived from a common sensory patch suggests an additional mechanism might exist to correlate morphogenesis and sensory induction (Wu and Oh, 1996; Torres and Giraldez, 1988). Although the mechanism regulating the main specification events remains elusive, there is now consensus that the axis specification and sensory organ formation involves multi-step regulation.

1.15.4 Cell lineages of the inner ear 1.15.4.1 The neurogenic lineage The delamination of neuroblasts from the otic vesicle represents the first cell lineage to differentiate. The potential to generate neurons and the identity of the neurons generated by the otic vesicle is not determined until at least at or after the placode stage (Vogel and Davies, 1993). Early expression of genes during the otic vesicle stage suggests that, although morphologically indistinguishable, some cells among the pseudostratified epithelium of the otic vesicle are already stepping into certain cell lineages. The neuroblasts in the epithelium of otic vesicle are already P tubulin positive (Torres and Giraldez, 1998). Another early trait of neuronal development within the undifferentiated epithelium is the expression of c-ret, a gene responsible for neuroblast determination in the gut. It was found to be expressed in a restricted area of the inner ear of chick (Robertson and Mason, 1995) and rat (MJ Lee, chapter 5). Veterbrate homologues of genes responsible for lateral inhibition are expressed in patches of undifferentiated epithelium, including the chick homologues of c-Delta, c- Notch and c-Serrate (Myat et al., 1996) and rat homologues of Jagged, Delta 1, Notch 1-3 (Lindsell et all, 1996). The c-Notch is expressed in the otic epithelium from

71 placode stages onward, while c-Delta-1 is expressed in scattered cells of the neurogenic region of the otic epithelium before the neural cells delaminate from the otic epithelium (Adam and Lewis, unpublished; see Whitfield et al., 1997). Based on these observations, a similar mechanism to the lateral inhibition regulated by notch- delta signalling in drosophila was proposed for the generation of the neuronal lineage of the inner ear (Whitfield et al., 1997)

1.15.4.2 The sensory cells: hair cells and support cells During the formation of the structure of the inner ear, in specific sensory patches the hair cells start to differentiate differently from the supporting cells. The molecular composition of the hair cells shares striking similarity to the eighth nerve neurons, at least in the cichlid fish (Presson, 1994). In the organ of Corti the nuclei of hair cells migrate upward and form a pseudo upper layer of nuclei above the nuclei of the support cells (Forge et al., 1997). In the bird the hair cells were regenerated by differentiating from the progeny of supporting cells that have been stimulated to re­ enter the cell cycle (Contanche et al., 1994). There is evidence to suggest that some supporting cells can convert to hair cells without cell division (Presson et al., 1996; Adler et al., 1997; Li and Forge, 1997). Evidence suggests that the hair cells and the support cells share a common precursor which may be a subset of support cells (Presson, 1994; Stone et al., 1996; Warchol and Corwin, 1996). Cell fate segregation in the sensory epithelium is still largely unknown. The notch-delta signalling mechanism used in the determination of the neuronal cell fate in the otic vesicle is believed to operate in the determination of neurogenic cells, as well as in the determination of the hair cells versus the supporting cells (Whitfield et al., 1997). Although hard data is still required to decide whether the notch-delta mechanism works in the finer tuning of the cell types within the sensory patch, another observation supports the idea that the maintenance of cell type and cytoarchitecture of the organ of Corti depends on the correct development of the hair cells. In the null mutation of BrnS.l mice, there is a complete absence of cells with hair cell characteristics (Erkman et al., 1996). Cells are present at the normal location of hair cells in the organ of Corti, but their nuclei are not well separated from the underlying supporting cells. By P14 the Deiter cells, pillar cells and other supporting cells are

72 either absent or indistinguishable, presumably secondary to the failure of development of hair cells (Erkman et al., 1996; Ryan, 1997).

1.15.4.3 The lineage of melanocytes In the inner ear melanocytes are distributed in the stria vascularis, modiolus, osseous spiralis lamina, subepithelial tissue of endolymphatic duct and sac, and dark cell areas of the utricle and crista ampullae (Conlee et al., 1989). Based on their location in regions of high metabolic and secretory activity (LeFerrier et al., 1974), and mutations linking defects in pigmentation with sensorineural deafness (Steel and Brown, 1994), melanocytes are believed to be the cell types responsible for maintaining the ionic difference between the endo- and epi-lymph in the stria vascularis and TV. In mice with defective melanocyte formation, hearing of mice is impaired due to the abnormal ionic composition of the endolymph (Motohashi et al., 1994; Steel and Brown, 1994). The source of these melanocytes is a subpopulation of migrating neural crest (Breathnach, 1988) . In splotch mice, with Pax-3 gene mutations, the defect in neural crest derivatives also affects the development of melanocytes in the inner ear and results in congenital deafness and patchy pigmentation (Auerbach, 1954). Immunochemical and ISH experiments using recently identified melanoblast markers such as MEBL-1, TRP-2/DT and mift directly showed that a melanogenic subpopulation of cranial neural crest migrates directly into the inner ear to their final destination (Kitamura et al., 1992; Steel et al., 1992; Nakayama et al., 1998), probably after the location of the sensory epithelium is specified.

1.15.4.4 Non-sensory, non-melanocyte epithelial cells Apart from the melanocytes, the nonsensory epithelium consists of cells of which the function (s) remain mostly unknown and the names for these cells are variable between literatures. These nonsensory epithlium cells are embedded in the modiolus bone, which is rich in capillaries (Spicer and Schultte, 1998; Sakaguchi et al., 1998). Following the opening of channels in hair cell stereocilia which generates electric stimuli, the excess is taken up by a sub-group of non-sensory epithelial cells and passes by the mechanism of ion exchange, to be recycled back to endolymph

73 (Schulete and Steel, 1994; Spicer and Schulte, 1994, a, b, 1996; Kikuchi et al., 1995). The is also actively transported from the perilymph in the scala tympani and scala vestibule by a chain of different types of fibrocyte in the lateral wall and spiral ligament back to the scala media (Spicer and Schultte, 1998; see figure 1-6). Apart from these ion balancing cells, the bone lining cells are another type of non- melanocytic cell which does not reside in the sensory epithelium in the inner ear (Chole and Tinling, 1994). They can be found in the blood-labyrinth barrier, an interface where the spiral ligament meet the capillaries and regulated exchange of blood and labyrinth fluid takes place (Suzuki et al., 1998). The ‘perilymph gusher’ phenotype, which is due to a mutation in the Brn-4 gene, is the only mutation reported to date that is directly related to a defect in the lateral wall (de Kok et al., 1995).

1.15.5 Factors responsible for the induction and differentiation of the otic vesicle In a study of the Salamander inner ear, Yntema (1950) showed that induction of the otic placode and vesicle depend largely on adjacent mesoderm while the differentiation of otic vesicle very much relies on the presence of rhombencephalon. Experiments on in vitro explanted otic primordia showed that formation of the otic vesicle from placodal or pre-placodal stages requires the rhombencephalon (Noden and Van de Water, 1986; Repressa et al., 1991). In transfilter experiments, it is suggested that age-associated diffuse factors from neural tube rather than cell-cell interaction is needed for induction and development of the otic vesicle (Van de Water et al, 1983). The importance of inductive effects exerted from hindbrain on the inner ear during development was also demonstrated by Van der Walter (1981). He showed that otic vesicle cultured from early embryos of E9 or 10 mouse are not viable without coculture of a piece of neural tube, while vesicles from older embryos lived well without CNS tissue and differentiated normally. Genetic analyses in mouse and zebrafish models have provided further evidence of the role of the neural tube in otic vesicle development (Deol, 1966; Steel, 1995; Whitfield et al., 1996; Malicki et al., 1996). The vast majority of the identified mutations affecting inner ear also showed hind brain malformation. It is proposed that defects in the ear are, in these cases, secondary to the hind brain mutation. Molecular and expression analysis of the

74 genes responsible for such phenotypes has confirmed that they are in fact expressed exclusively or principally in the neural tube. For example in the Splotch mutation, the neural tube was not closed and the otic vesicle was not in proper contact with the neural tube (Doel, 1966). Furthermore, mesenchymal cells are important in maintaining the gross morphology of the inner ear, after induction and initial axis specification. In an experiment in which E ll mouse otic vesicle was cultured in the absence of neural tube, otic vesicle developed in vitro into a semicircular duct, vestibule and cochlea, when the surrounding mesenchymal is intact, while removal of the ventral part of mesenchymal tissue resulted in cystlike extrusion of the cochlea duct (Li et al, 1978).

1.16 The nose 1.16.1 Terminology The olfactory nerves are accompanied by glia in both their PNS and CNS sections. As it is a fact that the CNS/PNS boundary is perforated by olfactory axons and glia, it is difficult to regard the olfactory ensheathing layer as belonging exclusively to the CNS or PNS. Therefore in this chapter ‘olfactory ensheathing layer’ has been used to indicate regions central to the apparent olfactory nerve and lateral to the glomerular layer. The glial cells residing in this area will be referred to as ‘olfactory ensheathing cells’ or ‘ensheathing cells’. ‘Central type glia’ will be used to indicate the astrocytes or oligodendrocytes which are believed to derived from the ventriciular or sub ventricular zone of the brain or spinal cord. I will use ‘olfactory Schwann cells’ for the glial cells which reside in the PNS portion of the nerve up to the olfactory ensheathing layer, while ‘olfactory glial cells’ will be used as a collective term for the ‘olfactory Schwann cells’ and the ‘olfactory ensheathing cells’ .

1.16.2 An overview of the olfactory system In the adult the sense of smell is processed within several distinct anatomical structures; the olfactory epithelium of the nose, the olfactory bulb of the brain, and the olfactory cortex. Inhaled molecules bombard the cilia and microvilli of the receptor neurons in the olfactory epithelium and by mechanisms still unclear this signal is translated into electrical impulses which propagate along the olfactory

75 nerves. The olfactory, or the first cranial nerve, consists of axons of the receptor neurons in the olfactory epithelium. It projects to the olfactory bulb and forms synapses with the secondary sensory neurons in the glomeruli of the bulb. Olfactory Schwann cells are found accompanying the olfactory nerves from the epithelium all the way to the olfactory ensheathing layer and in the CNS portion of the nerves (see origin and development of the olfactory Schwann cell). After several relays within the olfactory tract, these signals are processed and perceived as sense of smell in the sensory cortex.

1.16.3 Plasticity in the olfactory system 1.16.3.1 Neurogenesis in the olfactory epithelium Neurogenesis in the olfactory epithelium continues throughout the entire life of the animal, with the proliferative basal cell population of the epithelium serving as the stem cells (Graziadei and Monti-Graziadei, 1978a,b, 1979). Unlike other primary sensory neurons which innervate the CNS, a mixture of neurons of different levels of maturation are always found in the population of the olfactory receptor neurons. They can roughly be categorized as 1. the oldest cells that have already made synpatic contact with the neurons of the olfactory bulb, the CNS element of the olfactory pathway. 2. a group of immature neurons whose axons have not reached their targets. 3. The least mature cells which are newly committed to the receptor cell lineage and beginning to sprout their axons (Graziadei and Monti-Graziadei, 1978 a,b). The activity of neuronal death and regeneration of neurons occurs throughout adult life in the olfactory epithelium, in the absence of transection. The axons of new formed olfactory receptor neurons follow the same course of regeneration during axotomy as in the absence of transection, suggesting that it is the axonal growth of the most immature neurons that contribute to the neuronal regeneration and succesful connection in the CNS, not the regeneration of axons of older cells (Doucette, 1990), although there are reports that argue against this hypothesis (Cancalon, 1987). The fact that the moleuclar phenotype of the olfactory ensheathing cells is different when the olfactory nerves are transected probably is because that the axon associated with the olfactory Schwann cells belongs to the older neurons (Anders and Johnson, 1990).

76 1.16.3.2 The ability of olfactory axons to regenerate in the CNS The fact that olfactory receptor neurons undergo a continual process of replacement, which includes a continuous degeneration of old neurons and formation of new synapses within the CNS (Barber, 1982; Graziadei and Monti Graziadei, 1978; Monti Graziadei and Graziadei, 1979), makes the olfactory nervous system an useful model for the study of neuronal degeneration, regeneration and plasticitity. Regeneration in the olfactory system is different to the ‘axonal’ regeneration of anywhere else in the nervous system. As opposed to the constant growth and building up of connections to the CNS of the olfactory stem cells, other nerves are only capable of regrowth of the axonal part of the neuron when the neuron survives the transection. Furthermore, in the PNS, the regenerating axons often reinnervate the sensory and effector organs (Kiernan, 1979), while in the CNS the regenerated axons from the periphery often fail to cross the PNS/CNS TZ to establish connections with the CNS (Carlstedt et al., 1989; Liuzzi and Lasek, 1987; Stensaas et al., 1987). However, the axons of olfactory receptor neurons are capable of entry to and growth within the CNS in adult mammals (Barber, 1982; Graziadei and Monti Graziadei, 1978; Monti Graziadei and Graziadei, 1979). The factors which contribute to the successful growth of olfactory axons in the CNS are still unclear. Of several factors there were two of special interest: A. the immaturity of the olfactory neurons, B. the type of glia that ensheaths these axons within the nerve fibre layer of the main olfactory bulb. The molecular features of the regenerating olfactory neurons do not enable them to grow through every kind of CNS microenvironment, thus the glial type which resides in the pathway of olfactory axons might be important for the characteristic regeneration and synapse forming ability of the olfactory axon (Doucette, 1990). The olfactory ensheathing layer of the olfactory bulb, which is in the CNS/PNS TZ, is therefore of high interest.

1.16.4 The origin and development of olfactory Schwann cells The olfactory Schwann cells are derived from precursor cells in the olfactory epithelium (Marin-Padilla and Amieva, 1989; Chuah and Au, 1991), which in turn are derived from the olfactory placode (Constanzo and Graziadei, 1987; Hinds, 1972 a,b). In xenopus laevis the olfactory organs originate from paired thickenings of the cranial

77 ectoderm called the olfactory placodes. During the course of development, these placodes invaginate to form first olfactory pits and, ultimately, the olfactory cavities. The ectoderm of the olfactory placodes is divisible into two morphologically identifiable layers: the superficial nonnervous layer (epiblast) and the deeper nervous layer. The nervous layer of the olfactory placodes is continuous at early developmental stages with the layer that gives rise to the neural plate, which is the precursor of the entire CNS. The olfactory placodes comprise the craniolateral portions of the placodal thickening, which extends rostrally and laterally from the neural plate proper (Klein and Graziadei, 1983). The craniaolateral portion of the placodal thickening gives rise to olfactory organs (Constanzo and Graziadei, 1987). In the mouse the olfactory placode is recognized as a pronounced thickening of the ventrolateral ectoderm and is separated from the anterior part of the forebrain by mesenchymal tissue at ElO, the earliest stage examined (Hinds 1972a, b). As early as E l2 in mouse embryos, the nasal epithelium basal lamina is perforated at many sites by both existing axons and precursors of olfactory Schwann cells. The glial progenitors utilize the opening already made by the axon and remain in close association at all times with the latter during the migration to their targets in the telecephalon (Marin-Padilla and Amieva, 1989). Olfactory nerve fascicles, which consist of a group of densely packed small diameter axons enveloped by their glial cells, join together progressively into larger ones when moving toward the telecephalon. It should be noted that this topographical arrangement of the sheath cells and the axon bundles remains unchanged during the process of merging (Marin- Padilla and Amieva, 1989). Upon reaching the telecephalon, the olfactory nerves squeeze in under the meningeal structure and make direct contact with the surface of the telecephalon. The olfactory nerve perforates the olfactory bulb primordium first in individual or small groups of axons, followed by the penetration of their accompanying glial progenitor cells, and then the arrangement of the glomerulus, a unit consisting of the axon terminals and their glial cells, within the CNS. (Doucette, 1989; Marin-Padilla and Amieva, 1989). In spite of the glia accompanying axons into the CNS, most of the peripheral glial progenitor cells remain outisde the CNS and, together with the olfactory axons, form a marginal layer over the rostral wall of the invaginating olfactory bulb (Doucette, 1990)

78 1.16.5 The phenotypes of olfactory Schwann cells: a mixture of PNS and CNS phenotypes The notion that the peripheral glial progenitor cells differentiate not only into olfactory nerve Schwann cells but also into ensheathing cells of the central olfactory fascicles (Doucette, 1989; Farbman and Squinto, 1985; Marin-Padilla and Amieva, 1989), has made the olfactory glia an interesting subject in studying the regeneration of axons into the CNS. The olfactory glia possess a mixture of peripheral and central glial phenotypes (for review see Doucette 1990 and Devon and Doucette, 1992). These cells were considered originally more similar to astrocytes, because firstly, the major intermediate filament of olfactory Schwann cells is of central type glial fibrillary acidic protein (Barber and Dahl, 1987; Barber and Lindsay, 1982), which is is not expressed by peripheral Schwann cells (lessen et al., 1984; Jessen and Mirsky, 1984). Secondly, the olfactory Schwann cells are not individually enwrapped by their own basal lamina (DeLorenzo, 1957; Frisch, 1967). Lastly, the olfactory Schwann cells form a part of the glia limitans of the olfactory bulb and are in direct contact with astrocytes. The above features have led researchers to believe that these cells were more similar to astrocytes than Schwann cells (Doucette, 1990). Although when cultured in vitro, the molecular phenotype of these cells resembles the non- myelinating Schwann cells (Jessen et al., 1990), they cannot be induced to express Po or MBP by elevating the intracellular cAMP levels (Barnett et al., 1993), arguing that they were astrocyte-like cells.

However, several Schwann cell phenotypes have been associated with the olfactory Schwann cells. Olfactory Schwann cells are derived from the olfactory placode (Doucette 1989, Farbman and Squinto, 1985, Marin-Padilla and Amieva, 1989) and not from the subventricular zone of the developing brain, which is believed to be the source of the 0-2A progenitor cells (Yu et al., 1994). When cultured the olfactory Schwann cells express 217c antigen, p75NGF-R (Fields and Dammerman, 1985). They also share the feature of expressing the Ll/Ng-CAM cell adhesion molecule of non-myelinating Schwann cells. The expression of myelin protein Po recognized by 1E8 antibody in the olfactory Schwann cells in the chick (Norgren et at, 1992) and the ability to myelinate DRG neurons in culture (Devon and Doucette, 1992) made it

79 likely that the olfactory Schwann cells are more closely related in lineage to peripheral Schwann cells rather than astrocytes.

1.16.6 The lineage of olfactory ensheathing cells: a mixture of two cell lineages or two phenotypes of a same lineage? The controversy of the lineage of olfactory ensheathing cells was further fuelled by the discovery that, although the cell population of olfactory ensheathing cells was believed to derive from the olfactory epithelium, some cells of astrocytic phenotype were found in the olfactory ensheathing layer and were believed to be derived from the CNS (Doucette, 1989). Indeed the discrepancies of the lineage origin of olfactory Schwann cells could be rooted in divergent procedures of dissection and different understandings of nose anatomy. To get round this problem, Barnett and colleages (1993) purified olfactory nerve ensheathing cells using monoclonal antibody to 04 in conjuction with FACS cell sorting technique. Study of this purified population showed that these cells express both phenotypes of non-myelinating Schwann cells and type-2 astrocytes. A further study using more neural markers (Franceschini and Barnett, 1996) showed that these 04 enriched populations could be sub-devided into two phenotypes, one which has p75NGF-R immunoreactivity while the other expressed PSANCAM. The corresponding phenotypes can be found in vivo, with the p75NGF-R positive cell population always encompassing the PSANCAM positive cells. These two antigenic phenotypes could be induced in a clonal olfactory nerve ensheathing cell line, suggesting the two cell types share a common lineage (Franceschini and Barnett, 1996). Morphologically two types of cells could also be found in olfactory nerves as they progress toward the olfactory bulb (Doucette, 1989; Valverde et al, 1992). Judging from the location, they are suggested to correspond to the two distinct molecular phenotypes of the olfactory ensheathing cells (Franeschini and Barnett, 1996).

80 Immuno­ globulin domain

Extracellular

Cytoplasmic

Figure 1-1 The structure of Po protein. Immunoglobulin domains are indicated by half circle. The fibronectin type III repeats that are present in some members of the immunoglobulin superfamily are absent in Po protein. No phosphoinositol lineage to the plasma membrane is found. (Adapted from Lemke, 1992) luiuunmuiu Major dense line (cytoplasmic)

c m tnm nt^tn

Intraperiod line (extracellular) mmmt Myelin UIUUUI ^ Major dense line ^ -150 A (cytoplasmic)

nitnnimtmfm

Figure 1-2 Myelin membrane configuration of Po. This diagram illustrates the spatial distribution of Po protein in one-and -a-half 150-A repeat periods of the compacted myelin sheath. The immunoglobulin-related Po extracellular domain functions as a homotypic adhesion molecule to promote compaction at the intraperiod line, and the basic cytoplasmic domain promotes membrane adhesion at the major dense line. Electrostatic interaction between the positively charged proteins and negatively charged lipids of the apposing membrane is shown, (from Lemke, 1992) Migration j Derivatives

Figure 1-3

(top) Cell-marking techniques have revealed that trunk neural crest cells migrate along two pathways: a dorsolateral pathway (OLP) whose cells form pigment cells (PC), and a ventral pathway (VP) whose cells form the dorsal root ganglia (DRG), sympathetic ganglia (SYM), adrenomedullary cells (AM) and aortic plexuses, (bottom) In the rostrocaudal dimension, neural crest cells migrate through the rostral (R), but not caudal (C), half of each somitk sclerotome (Scl). However, their migration on the dorsolateral pathway between the ectoderm (Ec) and dermomyotome (DM) is unsegmented. NT, neural tube; No, notochord; Ao, aorta. Reproduced from Bronner-Fraser, 1993. ssc

es ES CL

ED MU' MN MS-

Vlllg ■ TV

BP

ML

Dorsal

Medial Lateral

Ventral

Figure 1-4 Gross structure of chick inner ear Specialized sensory patches contain hair cells surrounded by supporting cells. BP, basilar papilla; CL, lateral crista, CP, posterior crista; CS, superior crista; ED, endolymphatic duct, ES, endolymphatic sac, ML, macula lagena; MN, macula neglecta; MS, saccular macula; MU, utricular macula, SSC, semicircular canal, TV, tegmentum vasculosum. Adapted from Fekete, 1996. Scaia vestibuii

Stria vascularis

Scala media

Tectorial membrane

Spiral ganglion

Scala tympani Organ of Corti

Figure 1-5A A cross section of one turn of cochlear duct (reproduced from Steel and Brown, 1994).

R eissner Stria vascularis Membrane Tectorial m em brane

Supporting cells

Outer

Phalageal ""'^''Cell

acoustic nerves'ocy; y (Ph. Cells) Figure 1-58 Organ of Corti (adapted from Diamond et al., 1985) Medial Lateral

Scaia Vestibuii

Media

"füFlW;

Scala ly in p a n t^ ^

Figure 1-6 A schematic representation of the proposed medial and lateral transcellular routes for dispersal and conservation of K effluxed from inner and outer hair cells during auditory transduction. Ions released from inner hair cells (IH) diffuse medially through border cells (B’), inner sulcus cells (IS) and lateral interdental cell (LI) columns to the undersurface of the tectorial membrane and from ISCs through stellate fibrocytes (SF) to capillaries (Cap) or to central interdental cells (Cl) and the scala media. K+ pumped from scala vestibuii into supralimbal cells (SL) flows donwgradient to light fibrocytes (LF) and medial interdental cells (MI) for return to the scala media. In the lateral route, K+ effluxing from outer hair cells (OH) is resorbed by Deiter (D) and tectal cells (T) and flows via gap junctions through Hensen(H), Claudius (C), and outer sulcus cells (OS) and their root processes (RP) to efllux into stroma maintained at a low K+ level by the Na,K-ATPase activity of type II fibrocytes. K+ subsequently diffuses via gap junctions through type I fibrocytes (la, Ib) and striai basal (B) and intermediate cells (I) into the intrastrial compartment kept low in K+ by the pumping activity of striai marginal cells (M). K+ resorbed by type V fibrocytes from scala vestibuii diffuses downhill through Ib then la fibrocytes to the stria. B=basal cells: B’=border cell; Cap=capillary; C=CIaudius cell; CI=central interdental cell; D=Deiter cell; H=Hensen cell, I=Intermediate cell, IH=inner hair cell; IS=inner sulcus cells, M^marginal cell Ml^medial interdental cell; LF=light fibrocyte; LI=lateral interdental cell, OH=outer hair cell, OS=outer sulcus cell, RP^root process; SF=stellate fibrocyte; SL=supralimbal fibrocyte; T=tectal cell, la, Ib, II, IV, and V= types of lateral wall fibrocytes. Reporduced from Spicer and Schultte, 1998. Chapter 2 Materials and Methods

87 Materials Reagents for histochemistry Paraformaldehyde (FF) was purchased from Fluka (UK). Glutaradehyde and O C T compound (Tissue Tek), Araldite cy212, dodecenyl succinic anhydride (DOSA), dibutyl phythalate, benzyldimethylamine (BDMA) were from Agar Scientific Ltd (Essex, UK). TESPA (3-aminopropyltriethoxysilane), gelatin (type A from porcine skin), Poly-L-Lysine (PEL) solution, and methyl salicylate (oil of wintergreen) were from Sigma Chemical Company (Poole, UK). Polyester wax (Steedman, 1957) was from BDH laboratory supplies (Poole, UK). Alkaline phosphatase red substrate kit was from Vector Laboratories, Inc. (Burlingame, CA, USA).

Reagents for molecular biology Ultraspec RNA isolation kit was from Biotec Laboratories, Inc (Kinpton, Hertforshire, UK). Random hexamer and Superscript II reverse transcriptase. Tag polymerase, additional polymerases and RNase inhibitor were from Promega Coporation (Madison, USA). RNA ladder (0.24-9.5kb) was from GibcoBRL (Paisley, UK). QIAEX II kit for extracting cDNA from agarose gels, Miniprep and Maxiprep kits, were from Qiagen (Crawley, West Sussex, UK). Original TA cloning Kit for

cloning the PCR (polymerase chain reaction) fragment, which includes the pCR 2 .1 vector and T4 DNA ligase, was from Invitrogen Europe (Leek, Holland). All electrophoresis reagents were from Bio-Rad (Munich, Germany). SP6/T7 transcription kit for the DIG labelled riboprobe and additional NTPs were from Boehringer Mannheim (Lewes, UK).

cDNA pmcret 7 encoding a 2 . 8 kb fragment of the c-ret gene subcloned into Bluescript (SK) was a gift from Dr. V. Pachnis (Schuchardt et al., 1994). cDNA containing the whole sequence of mouse Dll gene (Bettenhausen, 1995) sub cloned in Bluscript (SK) was a gift from Dr. D Ish-Horowitz, and cDNA encoding l.Skb sequence of Serrate-1 (Jagged-1) (Mitsiadis et al., 1997) subcloned in Rvin pKs was a gift from Dr. G. Lewis, both with the kind permission of Dr. D. Henri que. cDNA encoding sequence of entire DM-20 subcloned in Bluscript (KS) was a kind gift from Dr. B. Zalc (Timsit et al., 1992). cDNA fragment Human ErbB3 bp 1250-3’UTR of

88 the ErbB3 gene subcloned in Bluescript was a gift from Dr C. Birchmeier (Meier and Birchmeier, 1995). The cDNA encoding the entire sequence of FGF-3 (int-2) gene sub cloned in pGEM3 was a gift from Dr. C. Dickson (Mansour et al., 1993). The cDNA encoding 750bp Pstl-Apal fragment of the Krox-20 gene subcloned in Bluescript (KS) was a gift from Dr . D. Wilkinson (Chavrier, 1988, 1990). The Krox- 20 DIG-riboprobe is transcribed by Dr. E. Parmantier and used with permission. cDNA encoding the Msx-1 (Hox-7) was a gift from Dr. K. Patel with kind permission from Dr. Hill (Robert et al., 1989) . A cDNA (SN63c) encoding the entire Po coding sequence ( 1 . 8 kb) (Figure 2-1) sub cloned in to pGEM4 was a gift from Drs. G. Lemke and 1. Griffiths (Lemke and Axel, 1985; Griffiths et al., 1989). cDNA encoding a 500 bp Dral fragment of the SCIP/Oct - 6 gene including the 3’ untranslated region subcloned in pSP71 was a gift from Dr. D. Meijer (Blanchard et al., 1996).

Reagents for ISH Proteinase K was from Fluka (Buchs, Switzerland). Form amide, sodium dodecyl sulphate (SDS), and Improved Aquamount (Gurr) were from BDH (Pool, UK). Yeast tRNA is from GibcoBRL (Paisley, UK). Heparin (sodium salt from porcine intestinal mucosa), and levamisole (L[-]-2,3,5,6-tetrahydro 6-phenylimidozo[2,l-b]thiazole), and Tween-20 (polyoxyethylenesorbitan monolaurate) were from Sigma Chemical Company (Poole, UK). Sheep anti-DIG antibody (Fab fragment), sheep anti- fluorescein antibody (Fab fragment), fluorescein conjugated UTP, DIG conjugated UTP, were purchased from Boeringer Mannheim (Lewes, UK).

Reagents for immunocvtochemistrv Lysine and sodium azide used for the antibody diluting solution and goat anti-mouse IgG- conjugated with horseradish peroxidase (Fab fragment) were from Sigma Chemical Company (Poole, UK). Fluorescein-conjugated goat anti-mouse Ig or goat anti-rabbit Ig, fluorescein-conjugated goat anti-mouse or goat anti-rabbit Igs, tetramethyl rhodamine-conjugated goat anti-mouse or goat anti-rabbit Igs, were from Cappel Organon Teknika Corp. (USA). Biotin conjugated sheep anti-mouse and sheep anti-rabbit antibodies, and streptavidin conjugated to fluorescein were from

89 Amersham International (Bucks, UK). The Vectastain Elite kit and ABC kit were from Vecta laboratories (Peterborough, UK). The Citifluor antifade mounting medium was from University of Kent, Canterbury, UK.

Rabbit anti-GAP-43 antibody was a gift from Dr. R. Curtis (Curtis et al., 1991). Rabbit polyclonal anti-Po was generated in the laboratory by Dr. L. Morgan (Morgan et al., 1994). Monoclonal antibody to the extracellular domain of Po was a gift from Dr J. J. Arcbelos (Arcbelos et al., 1993). Monoclonal antibody to MBP was from Boehringer Mannheim (Lewes, UK). Hoecbst dye H33258 (bisBenzimide) was from Sigma Chemical Company (Poole, UK). Rabbit antibody to SI00 and rabbit antiserum to GFAP, were supplied by Dakopatts (High Wycombe, Bucks, UK). Monoclonal mouse antibody 192 IgG, recognising the p75NGF-R (Taniuchi et al., 1986), was a gift from Dr. E. Johnson Jr. Monoclonal antibody TUJ-1, recognising the (33 isoform of tubulin (Moody et al., 1987), was a gift from Dr. A. Frankfurter. The CRABP antibody was a gift from Dr. M. Mad en.

Reagents for cell culture PEL for coating coverslips, selenium, transferrin, putrescine, thyroxine, triiodothyronine, insulin, cytosine arabinoside, L I5, and recombinant insulin were obtained from Sigma Chemical Company (Poole, UK). Calf serum was from Imperial Laboratories (Andover, UK). Foetal calf serum was from Advanced Protein Products (Brierley Hill, UK). Dulbecco’s modified Eagle medium (DMEM), Ham’s F12 medium, minimum essential medium (MEM) containing 0.002 M Hepes (MEMH), streptomycin, penicillin, amino acid solution, laminin, fibronectin and trypsin were from GIBCO Laboratories (Paisley, UK). Bovine serum albumin and glutamine were from ICN Biomedicals (Thame, UK). Collagenase (type2) was from Worthington Biochemical Co. (NJ, USA). The tissue culture plastic was from Falcon (Phillip Harris Scientific, London, UK). 13mm diameter coverslips were from BDH (Lutterworth, UK). IGF -1 was purchased from Kabi Pharmacia (Uppsala, Sweden). bFGF was from Pepro Tec Inc. (NJ, USA). Forskolin was obtained from Calbiochem-

Novabiochem (San Diego, USA).

90 Methods Unless otheiwise mentioned, the procedure ‘wash’ indicates incubation for 5 minutes, while ‘rinse’ means brief incubation for not more than 1 minute.

The embryos Sprague Dawley rats were used in all experiments. Dating of the embryos was described as in Christie (1965). The day when the vaginal plug appeared was counted

as embryonic day (E) 0. The mother was killed by CO 2 gas, and the uterus taken to L I5 medium on ice. The muscle was cut open, embryos transferred, and embryonic membranes carefully removed in LI5. The embryos were dated by their somite numbers and identified anatomical structures.

Axotomy operation Adult Sprague-Dawley rats weighing 90-110 gm, were anesthetized and the left sciatic nerve or the cervical sympathetic trunk (GST) was permanent transected. The proximal stump was re-routed into muscle, and the distal stump sutured at the end of cut site to prevent regeneration. The surgery was performed by Dr. K. R. Jessen with the appropriate licence from the Home Office. After surgery the rats were kept for 3,6 and 9 days before the cervical sympathetic trunk was removed for analysis. For

sciatic nerves the rats were kept for 6 weeks. The unoperated nerve on the right side was used as a control.

Dissection and fixation 4% PF in phosphate buffered saline (PBS) (Sambrook et al., 1989) was prepared by dissolving 4% PF at 65^C in hot Elga purified water containing a minimal amount of NaOH, and buffered with dilution of 1 OX PBS to IX. Fresh PF was used for ISH and kept no more than one week. For whole mount ISH, ElO, 11, 12 embryos were lightly fixed in PF for about 10 to 20 minutes, followed by dissection in PBS. The final dissection includes piercing the hindbrain and/or heart, if these structures are not relevant, and squeezing out the trapped blood in the dorsal aorta with forceps, followed by 4 hours of final PF fixation. After PBS washes and dehydration in graded

methanol/PBS (25/50/75/100) mixture, the embryos could be stored in -20^C in 1 0 0 %

91 methanol (see pretreatment for ISH). For sections, E14 to E17 embryos were pinned on silgard board with legs forming a 180 degree angle and perpendicular to the caudal-rostral axis to ensure that most sciatic nerve was included in the same section, before fixation in PF for 6 hours. The embryos were washed thoroughly in PBS, before proceeding to further embedding and sectioning methods (see embedding and sectioning ). For teased nerve preparations, the epineurium of nerves was removed and the nerves were cut into 1-5 mm segments. The packed bundles of nerve fibres were loosened first in PBS, followed by separating individual fibres of the moist bundles on gelatin slides. The individual fibres adhere to the gelatin surface as the PBS dries out. The teased nerves on slides were air dried for a further 10 minutes to ensure attachment. For in situ hybridization the teased nerves were then fixed in 4% PF for 2 hours followed by dehydration by washing in graded (30/50/70/80/95/100%) ethanol, 1 minute each. They were rehydrated with graded ethanol as above before further processing. For guts from various ages, different dissecting strategies were used. At E ll it was not possible to separate the visceral system from the adjacent structures. Therefore to examine the migrating population in the gut area, whole embryos were processed for ISH followed by cryosection or araldite sectioning (see below). For guts from E l2-14 embryos, the embryos were fixed in 4% PF for 4 hours and dehydrated, as for preparation for whole mount ISH (and could be stored at this stage), and rehydrated in PBS. The embryos were decapitated, leaving as much neck as possible. The rib, heart and liver were removed first, followed by separating the gut and dorsal aorta from the back. The oesophagus, stomach, intestine and colon could be separated as a whole piece with ease from other structures. It is possible to dissect the nodose ganglion at E l2, while this is not possible later on due to growth in the pharyngeal area. The guts were washed in PBS, refixed in 4% PF for 4 hours, dehydrated in a mixture of methanol in PBS (25/50/75/100) and stored in methanol at

-20°C. Guts of rats older than E l 8 were dissected out without prior PF fixation. The cavity of the dissected gut was cut open, residual food discarded, and visceral wall washed in PBS. Layers of visceral walls were pinned together on silgard board and fixed in PF for 6 hours, followed by a thorough PBS wash. They were dehydrated and embedded in polyester wax (see below). In some case of whole mount ISH, Ell embryos were cut into 6 segments, each 6 somites in length. The ectoderm and some

92 mesenchymal tissue were removed by collagenase digestion and dissection (for details see Chapter 4).

Embedding and sectioning Distortion of morphology often results from the embedding and sectioning methods used. Embryonic tissue is especially problematic because of its high water content and high proportion of mesenchymal cells. Several factors contribute to inadequate morphology. When the combination of fixation and embedding fails to protect and solidify the tissue, in the worst case it is difficult to cut sections, since the sections collapse. In other cases it is possible to compromise by using thicker sections but these give less cellular resolution. With methods which involve heavy dehydration, because of a need not to damage the antigen or mRNA of interest, a compromise is often made in the choice of fixative and thus results in distortion of mesenchymal tissue and separation of individual tissue compartments. The latter is a serious problem, since the tissues of interest in this study are mesenchymal. I here list several which gave good morphology and more cellular detail. Some methods do not give the best morphology but were used in cases when different antigens or mRNA needed to be compared in the same embryo.

1. Polyester wax methods This method was used for most of the ISH except in the studies of migrating neural crest, and for immunostaining of antigens which could endure long alcohol washes. In general this method gives good morphology and was used when possible. Whole embryos or nerves were fixed in 4% PF dissolved in O.IM sodium phosphate pH 7.2 overnight at 4^C. The next day the tissue was dehydrated through a series of graded alcohols: 50%, 70%, 90%, and three changes of absolute alcohol. For ages El 3-16 the time for each stage was 45 mins and for E l7-19, 75 mins. For nerves it was 30 minutes. Melted polyester wax solutions diluted in ethanol at 40^C are made in the following concentrations: 50%, 75% and 2 lots of 100% wax. The tissue is incubated in each of these in turn at 40^C for at least 1 hour or until the tissue sinks. The wax is changed to the last 1 0 0 % wax incubation and left to incubate at 40^C overnight to ensure equilibration. For sectioning the block was trimmed to small size. The wax

93 block is cooled with circulating cold water using a thermoelectric machine, Pelcool) before and during cutting. 7-lOuM sections were cut and mounted on PLL-gelatin coated slides (see below) in double distilled water on ice. The sections were dewaxed with 2 changes of 1 0 0 % ethanol, or until the sections looked clear, followed by an essential step of air drying to ensure adhesion. After rehydration through graded ethanol, 1-2 minutes per wash, the sections could be processed for ISH or immunostaining.

2. Fresh cryostat sections

This method was used when 2 different fixations were used on adjacent sections. It is suited to embryos as young as E l4. Rat embryos younger than this were very difficult to cut using this method. Embryos or tissue were embedded in powdered dry ice after excess fluid has been removed. They were attached to the cryo-stage with O C T compound, cryocut at 10- 14 p.M and mounted on gelatin coated slides. Alternatively, the tissue was embedded in O.C.T first. It was placed in a square foil boat or plastic mould in O.C.T compound, and frozen in liquid nitrogen cooled isopentane. Sections were thawed onto gelatin coated slides for immunostaining, or TESPA coated slides for ISH, air dried, and fixed in a specific way for some antigens, or in 4% PF in PBS for 2 hours for ISH. For ISH, at the end of the fixation the sections were washed twice in PBS, and dehydrated through graded ethanol, 1 - 2 minutes each, and air dried. They could be stored dessicated at -70^C. For cryosections and teased nerves, care should be taken to seal the sections in bags or leave dessicating agent in the storage box to keep the samples dry. They are rehydrated through graded ethanol to proceed with ISH.

3. Sucrose protected, gelatin embedded cryostat sections This method preserves good cellular morphology of young embryo and neonatal tissue (El 1 to P5). Because the gelatin is often contaminated with RNAses, this is not used as a method to obtain sections for ISH. It is used for post whole mount ISH sectioning rather than the polyester wax method since it does not wash away the blue- purple colour precipitation. It is also used for immunostaining of antigens which cannot endure excess alcohol washes. The tissue is fixed first, and the fixative used

94 depends on the antigen. 4% PF, methanol or ethanol at -20^C, 2% HCl, etc. were used for various antigens (see Immunocytochemistry). After thorough washes with PBS, the tissue was transferred to O.IM phosphate buffer, equilibrated for several hours or overnight, followed by incubation in 15% sucrose in O.IM phosphate buffer, until the tissue sank. It was changed to 7.5% gelatin plus 15% sucrose in phosphate buffer overnight in 37°C. The tissue was embedded in a square mould in gelatin solution. The solidified block were then gently frozen in liquid nitrogen cooled isopentane and sectioned. For embryos younger than El4 I used a -20 to -18 setting on the cryostat machine. The sections were thawed on to suitably coated slides for immunochemistry, or dehydrated through graded ethanol, air dried and stored for ISH.

4. Sucrose protected This method gave good morphology for young embryos (El 1 and 12) and does not 1. cause shrinkage and distortion between the neural tube and neural crest derivatives 2 . use gelatin which contaminates the ISH. The fixation of the tissue is the same as above. After rinses in PBS and incubation in O.IM phosphate buffer, the embryos were incubated in 30% sucrose in 0.1 M phosphate buffer until the tissue sank. The embryo is placed in OCT compound and frozen and sectioned as above. The collection of sections and pretreatment for immunochemistry or ISH is similar to that described in 3.

5. Araldite embedded semi-thin sections This method was not used as a method to obtain sections for ISH and is used for post whole mount ISH sectioning. The signal in whole mount embryos subjected to ISH was overdeveloped in the colour solution overnight. Colour precipitation trapped in the cavities was washed out as much as possible with PBT, with the aid of localized pressure generated by blowing PBT through a Pasteur pipette. Embryos were then fixed in 4% PF overnight, washed in PBT and dehydrated through 30,50,70 and 95% alcohol in a minimum time, i.e., the time for the embryos to settle in solution plus 1 minute. After 2 changes in absolute alcohol, the solution was replaced with methyl salicylate. Embryos were allowed to settle before another change of solution. They

95 were further incubated in a mixture of 50%/50% methyl saiicylate/araldite resin for 30 minutes, and left in neat araldite overnight with slow rotation. The resin was changed twice, each incubation was several hours, and embryos were embedded in fresh araldite. The block was placed in an oven overnight or until completely hardened. The block was sectioned at 4 p,M or less on a glass knife microtome. The sections were mounted on plain glass slides in water on a hot plate. To make the araldite resin, lOg aralditecy212, lOg dodecenyl succinic anhydride (DDSA), 0.8g plasticizer dibutyl phythalate, were mixed on a hot plate until clear, then 0.4 ml benzyldimethylamine (BDMA) was added. The mixture was left stirring for another

2 minutes and used while hot.

Coating of slides Cryosections adhered well to gelatin coated slides and withstood the immunochemical procedures. However, sections of polyester wax do not adhere well to the gelatin coated slides. Several coatings were tested, including gelatin only, PLL only, gelatin first then PLL, PLL followed by gelatin. The final choice was an initial PLL coating followed by gelatin. The slides were coated with high molecular weight PLL (0.1% w/v in distilled water) for 1 minute, dried in room temperature, and then coated with 1% gelatin with 0.25% chrome alum (room temperature). They were dried at room temperature. The PLL-gelatin coating melted when the hybridization temperature was over 65°C. In this case, Tespa coated slides, among other coatings tested, gave the best adherence. The glass slides were washed in acid alcohol (1% concentrated HCl in 70% ethanol), followed by washing in running tap water for 1 hour. The slightly alkaline tap water neutralises the previous acid wash. The slides were further washed 30 minutes in Elga water and 10 minutes in acetone, allowed to air dry and baked at 180^C for 2 hours. After cooling the slides were washed through the following reagents: 1. 2% TESPA in acetone 2. acetone 3. acetone 4. autoclaved Elga water and dried overnight at SO^C. The coating was activated in 2.5% (v/v) glutaraldehyde (2.5 ml of glutaraldehyde solution in 100 ml of PBS) for 1 hour. This was followed by 3 washes in autoclaved Elga Ultra pure water, 20 minutes each. After air drying these slides could be kept at room temperature or in the refrigerator.

96 RT-PCR (Reverse transcripase polymerase chain reaction) Total RNA was made from whole embryos at E ll, and freshly dissected sciatic nerves from El4 and P4 rats, using the ultraspec RNA isolation reagent. The concentration and purity of RNA extracted was measured in a spectrophotometer. The quality of RNA was checked by running a 2% agarose gel. Two prominent and distinct bands of ribosomal RNA, with the 28s 3 times as intense as IBs, implied a good extraction. 500 ng RNA was reverse transcribed to cDNA using random hexamers as primers and Superscript II reverse transcriptase as recommended in the manufacturer’s protocol. Equal volumes of the cDNA were used for PCR with the oligonucleotide primer pairs complementary to Po exons 4 and 6 (Brunden et al., 1992). The sequence for the synthesized oligonucleotide primer that is complementary to a sequence within exon 6 of Po cDNA is (5’- TTGGTGCTTCGGCTGTGGTC-3’). The upstream oligonucleotide primer that is complementary to a sequence within exon 4 is (5 ’ -TGTTGCTGCTGTTGCTCTTC- 3’) (Lemke et al., 1988). Each cycle was 94^C 1 minute, 52^C 1 minute, and 72^C 1 minute. In some experiments the annealing temperature was set to 60°C instead of 52^C. The reaction was run for 42 cycles to detect the low level at El 1. One-fifth of each reaction was electrophoresed on a 2% agarose gel cotaining 0.5|ig/ml ethidium bromide. DNA standards were generally included on gels.

Preparation of the constructs for ISH lp .1 of plasmid solution for different probes obtained from various laboratories was dried on Whatman No. 1 filter paper and transported or stored for up to 2 weeks. The plasmid was grown and cDNA purified using Qiagen miniprep and maxiprep kits according to the manufacturer’s instruction, and basic molecular biology methods according to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989). At least 4 clones were grown from the same plasmid. The cDNA was collected using the miniprep kit, the inserts excised with appropriate restriction enzymes and examined on a 2% agarose gel, before preparing the maxiprep. For the BMP-4 riboprobe, a cDNA corresponding to nucleotides 707 to 1275 was reverse transcribed with primer pairs complementary to the published BMP-4 sequence (Gene bank) from mouse E l2.5 heart and lung cDNA library (kind gift of Dr E. Parmantier). The cDNA was

97 electrophoresed on a 2% gel, and extracted from the agarose gel using a QIAEX II kit, according to the manufacturer’s directions. The cDNA was then sub-cloned into PCR2 plasmid using Original TA Cloning Kit according to the manufacturer’s instructions. The orientation of the insert was checked by enzyme digestion and electrophoresis on a 2% agarose gel. In vitro transcription reaction using plasmid containing 1250 bp human ErbB3 and 3’ untranscribed region (3’ UTR) gave no or very low amount of riboprobe, probably due to the nature of the construct and therefore the low efficiency of the RNA polymerase. The fragment between the two Xhol restriction sites was deleted by enzyme digestion, and the rest of the plasmid jointed at the Xhol sites by religation reaction using T4 DNA ligase. Plasmids containing the ErbB3 construct without the Xhol fragment (ErbB3-XhoI) were selected by preparing miniprep plasmid DNA from clones and checking the excised insert size by electrophoresis. Maxiprep was prepared for the good clones. The deletion of the Xhol fragment helped the transcription and riboprobe was prepared from the ErbB3-XhoI plasmid.

ISH Preparation of riboprobe The plasmid containing Po cDNA was linearized from the Clal site as a template for transcription of antisense probe. Xbal restricted enzyme used to generate a template for the probe was transcribed in the sense orientation. The linearized DNA to be transcribed was purified by phenol/chloroform extraction and ethanol precipitation.

After being resuspended in TE buffer (10 mM Tris, ImM EDTA, pH 8 ), the concentration of cDNA was determined by reading the optical density at 260 nM in a spectrophotometer. The DIG labelled riboprobe was transcribed using Boehringer Mannheim SP6/T7 transcription kit according to the manufacturer’s instructions. Restriction sites and polymerase used to generate riboprobes are listed in table 2-1. The transcription efficiency was checked by dot blot and spectrophotometer. The size of the probe was checked by agarose gel electrophoresis. A rough estimation of probe concentration was also made by comparing the density of gel band to the control DIG-labelled RNA from the transcription kit. The concentration of transcribed RNA cDNA was determined by reading the optical density at 260 nM in a

98 spectrophotometer. To achieve higher sensitivity, some probes were hydrolysed to a size of 0.1 kb by incubation in O.IM carbonate buffer (80 mM NaHCOg ,120 mM

Na 2 C 0 3 , pH 10.2) at 60° C. The incubation time required depends on the probe size (Digestion time = [initial kb-desired kb]/ [ 0.11 x initial kb x desired kb] [in minutes]). This yielded significant improvement in signal for the Po probe. On the other hand the ErbB3-XhoI worked better using a non-hydrolysed probe.

Prehybridization treatment Different prehybridization procedures were adapted for sections, teased nerves, whole mount embryos and cultured cells. They are described below.

Sections and teased nerves As described in Embedding and Sectioning, after storing, the sections were rehydrated in graded ethanol, followed by 2 washes in PBS. This was followed by proteinase K treatment in TE buffer (lOmM Tris and ImM EDTA , pH 8 ) for 5 minutes in room temperature. 10 pg/ml of proteinase K was used for sections and 1 p,g/ml for teased nerves. The tissue were refixed with 4% PF for 20 minutes after two washes in PBS. To reduce the non-specific electrostatic binding of the probe to the amino groups, sections and teased nerves were further treated with acetic anhydride

(0.25% in O.IM triethanolamine-HCL, pH 8 ) for 10 minutes. The tissue were dehydrated in graded ethanol and air dried before hybridization.

Whole mount embryos After dissection, the embryos were fixed in 4% PF in PBS (pH7) for 4 hours and were dehydrated through graded methanol in PBT (PBS with 0.1% Tween-20) . They could be stored in methanol at -20°C, or used immediately. The embryos were rehydrated reversibly through graded methanol in PBT, 75,50,25% respectively, 5 minutes twice for each wash. They were washed in PBT twice, and treated with 10 pg/ml of proteinase K in PBT at 37°C for 15 minutes. After refixing in 4% PF for 20 minutes and thorough washes in PBT, the embryos were prehybridized in hybridization buffer (50% form ami de, 5X standard saline citrate [SSC; pH 4.5], 1 %

99 sodium dodecyl sulphate [SDS], 50|ag/ml yeast tRNA, and 50p,g/ml heparin) at 70°C for 1 hour before incubation in hybridization mixture at 70°C overnight.

Cultured cells The cultures were fixed for 20 minutes in 4 % PF. For the neural crest cultures, the neural tube was removed 1 or 2 days after plating with fine needles. In some cases the neural tube was kept in place. To fix the neural crest cultures, an equal volume of 4% PF was added to the culture medium, left for 5 minutes, before further fixation for 20 minutes with fresh 4% PF at room temperature. The residual fixative was washed thoroughly in several changes of PBS. The cell membrane was permeablized by incubation in 1% Triton-100 in PBS for 30 minutes. Proteinase K was not used to avoid loss of cells from the substratum. The cells could be stored at 4^C in PBS. For in situ hybridization, the cultures were washed several times with PBS. Dehydration to 70% or 100% ethanol gives less satisfactory results.

Hybridization The riboprobes were diluted 1:200 in hybridisation buffer. Sections and teased nerves were hybridized in 30 to 100 |il hybridization mixture drop on each slide. The mixture was covered by a layer of plastic membrane cut out from new plastic bags and the slides placed in a sealed box moisturised by 4x SSC in the preheated oven. The coverslips were hybridized face down on a clean glass slide with a drop of 20 p,l hybridization mixture, and the slides placed as above. The embryos were prehybridized in Scotting’s buffer (see below) for 1 hour, and hybridized in hybridization mixture in a 7 ml plastic bijou in a preheated water bath. The following protocol was adapted from Rex and Scotting (1994) (referred to as Scotting’s method).

For the post-hybridization washes, the embryos were washed in Scotting’s buffer 1 (50% deionized form amide, 5X SSC, pH 4.5, 10% SDS) twice at 65°C, three times in buffer 2 ( 50% deionized formamide, 2X SSC) at 65°C, and three times in TBST (140 mM NaCl, 25 mM Tris-HCl [pH 7.5], 5 mM levamisoie, 0.1% Tween-20) at room temperature. The background colour was blocked with 1% fat-free milk in TBST for

100 1 hour, before incubation in alkaline phosphatase-conjugated sheep anti-DIG antibody diluted 1:2500 in 1% milk in TBST overnight. Before the colour reaction, the tissued were washed thoroughly in PBT overnight, 20 minutes in TBST with 1-2% Tween-20 and 3 mM levamisole, followed by 10 minutes wash in NTMT (100 mM Tris-HCl,

100 mM NaCl, 50 mM MgCl2 , pH 9.5) before incubation in the colour solution (150 jug/ml nitroblue tétrazolium salt and 75 |ig/ml 5-bromo-4-chloro-3-indolulphosphate, toluidinium salt, 100 mM Tris-HCl, 100 mM NaCl, 50 mM M gCy in the dark until optimal colour developed. Excess background color could be prevented if samples were washed in PBT with 1% Tween-20 overnight. The reaction were stopped by washes in TE buffer and postfixation with 4% PF. Sections, teased nerves and cultured cells were mounted in aqueous mountant (BDH Gurr). Whole mount embryos were photographed in PBT, or changed through graded glycerol in PBT and photographed in 80% glycerol in PBT (see also notes for photographing the whole mount tissue). For further analysis of whole mount tissue, the tissue was left in colour solution overnight to over-develop the colour precipitation. After this the tissue was fixed in 4% PF for at least 1 hour or longer depending on the size of the tissue. It was washed thouroughly with PBT, and embedded. Sucrose protected gelatin blocks sectioned to 10 to 14 pM were used most of the time, and 20 pM sections were used for Nomarsky optics (see photography). Araldite embedded sections were used when cellular resolution was required and the signal to noise ratio was good (see notes on photography).

For simultaneous detection of other markers in the ISH treated tissue, the primary antibody was included in the same incubation as the anti-DIG antibody, in 1% fat-free goat milk powder in buffer 1 (100 mM Tris-HCl and 0.15M NaCl, pH 7.5). After the colour reaction, the sections were rinsed in PBS, and incubated in the biotinylated secondary antibody for 30 minutes in antibody diluting solution (ADS) (see following), and visualized with streptavidin coupled with fluorescein, 1:100. The sections or teased nerves were post-fixed with 4% PF for 20 minutes, followed by Hoescht staining, before mounting in Citifluor.

Immunocytochemistry

101 Antibodies were diluted in antibody diluting solution (ADS) (PBS containing 10% CS, O.IM lysine and 0.02% sodium azide) unless otherwise stated.

GAP-43 Rabbit anti-GAP-43 antibody was used at a dilution of 1:100 overnight ADS, or in the case of post-hybridization staining, in 1 % skimmed milk (dried powder) in antibody dilution solution. It was followed by biotin conjugated sheep anti-rabbit antibody 1:100 for 30 minutes then streptavidin coupled with fluorescein 1:100 for 30 minutes.

Po The antiserum was purified by incubating with chloroform-extracted new-born rat skin for 48 hr, followed by precipitation with caprylic acid, and then 40% ammonium sulphate precipitation and dialysis by Dr. A. Brennan (Stewart et al., 1996). Cells were fixed in 2M HCl for 15 minutes, followed by extensive washes in PBS, then O.IM sodium borate solution for 10 minutes. The cells were blocked for 2 hours with ADS, washed, and then incubated in antibodies to Po overnight. A dilution of 1: 1000 was used for detection of the higher level of Po found in myelinating Schwann cells, and 1:200 was used as a sensitive method to detect lower basal levels of Po Antibodies were visualized with fluorescein-conjugated anti-rabbit Igs. Monoclonal antibody to the extracellular domain of Po (Archelos, 1993) was used at 1:50, followed by incubation in biotin conjugated sheep anti-rabbit antibody 1:100 for 30 minutes then streptavidin coupled with fluorescein 1:100 for another 30 minutes.

MBP

Monoclonal antibody to MBP was used at 1 : 100 in ADS, or in the case of post­ hybridization staining, in 1 % fat-free goat milk in antibody diluting solution. It was followed by biotin conjugated sheep anti-mouse antibody 1:100 for 30 minutes then streptavidin coupled with fluorescein 1 .TOO for 30 minutes.

Hoechst staining

102 Hoechst dye H33258 (bisBenzimide; Sigma) was used atl |Lig/ml in PBS for 10 minutes after PF fixation and prior to mounting the sample in Citifluor.

SlOO

Rabbit antibody to SlOO was diluted 1 : 2 0 0 in ADS. It was visualized with fluorescein-conjugated anti-rabbit Igs.

NGF-R Monoclonal mouse monoclonal antibody 192 IgG, recognizing the p75 NGF receptor, was used at 1:100 in ADS, followed by fluorescein-conjugated anti-mouse IgG.

CRABP The CRABP antibody was used at 1:400, and was visualized with biotin conjugated goat anti rabbit antibody followed by streptavidin/fluorescein.

TUJ-1 (section and whole mount) Monoclonal antibody to beta tubulin type 3 was diluted to 5 |ig/ml in PBS containing 2g/l gelatin and 1% triton. For cultured cells it was visualized with fluorescein- conjugated goat anti-mouse Igs. For staining on the whole mount embryo, the

endogenous peroxidase was inactivated with 1% H 2 O2 in PBT overnight. The embryos were first washed twice with TBS (50 mM Tris-HCl, pH 7.5, and 0.85% NaCl), 30 minutes each, and blocked with a mixture of calf serum: DMSO (4:1) plus 0.1% Triton-100 overnight. Antibody to beta tubulin type 3 was used 1:200 in the blocking solution overnight. Between the first and second antibodies, the embryos were washed with TBS for at least 5 hours, with changes every hour, then were incubated in goat anti mouse IgG conjugated with horseradish peroxidase. Fab

fragment, 1:200 in 1 % calf serum in PBS overnight. The embryos were washed thoroughly overnight in TBS, and incubated in 1 mg/ml DAB (3,3’-

diaminobenzidine) solution containing 0.02% H 2 O2 for up to 1.5 hour. The reaction was stopped by incubation in mixture of TBS: methanol (1:1) for 20 minutes.

GFAP

103 Rabbit antiserum to glial fibrillary acidic protein, GFAP, was used at a dilution of 1:100 in antibody diluting solution. It is revealed by indirect immunostaining with biotin conjugated sheep anti-rabbit antibodies followed by streptavidin conjugated to fluorescein.

About double staining The two primary antibodies were applied at the same time in ADS unless otherwise stated, or when combined with ISH, in 1 % dry milk powder in Buffer 1 (100 mM Tris-HCl and 0.15M NaCl, pH 7.5) with anti-DIG antibody. The antibodies were visualized with first incubation of biotin conjugated sheep anti-rabbit antibody followed by streptavidin-fluorescein, and cy3-conjugated goat anti-mouse Igs, respectively. On some occasions rabbit-biotin/streptavidin-cy3, and mouse- biotin/streptavidin-fluorescein, were used to amplify the signal for the monoclonal antibody. For details of double staining for in situ hybridization coupled with immunostaining see individual chapters.

Cell culture Defined medium The defined medium (DM) used in this study was a modification of the medium of Bottenstein and Sato (1979). Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium mixed 1:1 and supplemented with bovine serum albumin (0.3 mg/ml), transferrin (100 p.g/ml), glucose (7.9 mg/ml), putrescine (16 jig/ml), progesterone (60 ng/ml), thyroxine (400 ng/ml), triiodothyronine (10.1 ng/ml), selenium (160 ng/ml), dexamethasone (38g/ml)), glutamine (2 mM), penicillin (100 lU/ml) and streptomycin (100 lU/ml).

Preparation of coverslips and substratum coating 13 mm diameter coverslips were sterilized by baking at 140^C for 4 hours. They were incubated with 1 mg/ml PEL (MW>300K) for 24 hr at room temperature, washed over 3 days with 8 changes , then allowed to dry. They were kept cool and dry for at least 1 month, then coated with 1 |ig/ml of laminin in Dulbecco’s modified Eagle’s medium (DMEM) for Ihr in room temperature (RT) before use. The volume of

104 laminin was 10 |il for Schwann cell precursors and 20 pi for cells of other ages. For neural crest culture, the PLL coated coverslips were further coated with 25pg/ml flbronectin in alkaline DMEM for 30 minutes. These PLL-fibronectin coated coverslips were incubated in medium required for the experiments prior to plating.

Preparation of neonatal Schwann cells Schwann cells were prepared using an adaptation of the method by Brockes et al. (1979). Sciatic nerves were dissected from rats of different neonatal ages, the epineurial sheath removed and chopped into small pieces prior to enzyme digestion. The nerve pieces were incubated in a mixture of 0.25% trypsin, 04% collagenase in

DMEM at 37^C and 5% C 0 2 / 9 5 % air for 35 minutes. The tissue was further dissociated by triturating through a 200 pi pipette tip. The digestion was stopped by a dilution of 7 ml defined medium (DM). The cells were centrifuged at 1000 rpm at 4^C for 10 minutes, supernatant discarded, and pellet resuspended in DM containing 1 nM insulin. The cells were counted and plated to PLL-laminin coated coverslips and left for 3 hours at 37^C in 5% C 0 2 / 9 5 % air before topping up with medium, or fixing with fixatives.

Preparation of adult cervical sympathetic trunk (CST) cells Cervical sympathetic trunks (CST), of which 99% of the cells are non-myelinating Schwann cells, were taken from adult rats aged more than 4 weeks. The trunks were dissected, desheathed and chopped as for neonatal cells, and dissociated in mixture of trypsin and collagenase as above for Ihr. The tissue was triturated after 30 minutes before further incubation, and after a total of 1 hour incubation, through a 2 0 0 pi pipette tip. The cell suspension was diluted with 7 ml DM to stop the enzyme reaction, the cells centrifuged and resuspended in DM containing 1 nM insulin. The cells were counted and plated as described above.

Preparation of neural crest culture Neural crest cultures were prepared using a method adapted from Smith-Thomas and

Fawcett (1989) and Bannerman and Pleasure (1993). The most caudal 6 to 8 somites and 2 somites worth of continuous non-segmented plate region of El 1 embryos were

105 used. Segments were incubated in 0.1% collagenase in L I5 solution without calcium and magnesium (12 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1 ImM glucose, 1:20 amino acid solution buffered with NaHCOg) on ice for 15 minutes and 1 hour at 37^C. The tissue was transferred to LI5 medium containing 3ng/ml bFGF and 50 ng/ml IGF-1. The neural tubes were cleaned of surrounding somites by pipetting through 200ul pipette tips and by teasing with fine needles. The tubes were transferred to DM without dexmethasone containing 3 ng/ml bFGF and 100 ng/ml IGF-1, and plated in the same medium on PLL-fibronectin coated coverslips. Extra care was taken to maintain the CO 2 level and the temperature of the culture medium during medium changing and handling the tissue.

Notes on photography of whole mount tissue Two parameters, i.e., the signal to noise ratio of the color product and the degree of transparency of the tissue, were keys to the succesful photography of a whole mount tissue. Several protocols were developed to improve the signal to noise ratio of the color product, including 1 bleaching the embryo with 0 . 1 % H 2 O 2 , during the prehybridization step. 2. lowering the concentration of Tween-20 in the color solution. 3. raising the concentration of NBT/XP in the color solution. 4. increasing the Tween-20 (or other suface activant) concentration in the post-color solution washes. 5. washing of trapped color solution from the cavity with flushes of PBT. If the signal to noise ratio of the color product was good, the whole mount tissues were photographed in PBT in plastic dishes, or in PBT on top of a thin layer of 1% agarose gel, which gives an interesting blue background. Otherwise they were changed through graded glycerol in PBT and photographed in 80% glycerol in PBT, or dehydrated with ethanol, and changed through a graded mixture of ethanol and methyl salicylate and photographed in 100% methyl salicylate. The later method increases the transparency very efficiently, but the color fades away rather fast and the methyl salicylate is volatile and carcinogenic. 2:1 mixture of Benzyl benzoate/benzyl alcohol clearing was tried but in my hands did not significantly increase the transparency. Lighting is another way to increase the transparency. Dark field photography gives better transparency to the tissue than white field photography. Strong light from underneath increases the transparency, especially for those tissues

106 with a less than ideal signal to noise ratio. Side light gives more details of the tissue when the signal to noise ratio is good, but generates a cloudy effect when the ratio is bad.

Photography

For photographing the ISH of whole mount embryos, a Leica MZ 8 steriomicroscope equipped with a Sony video camera feeding digital images to a Mackintosh Powermac computer was used. The microsocope was lighted by a Zeiss ring illuminator with helium bulb, and the digital image was captured with a image grabber and processed with Adobe Photoshop 4 software. For everyday recording the image was printed using a Sony Printer, and for publication quality the image, after editing with Adobe photoshop, was saved onto an optical disc and emailed to the departmental printing facility. Some pictures were taken with a Leica MZ 6 Steriomicroscope equipped with a Leica photographic system (Courtesy of Dr Paul Martin ) using Kodak Ektachrome 64 Tungsten positive films. Smaller pieces of whole mount tissue such as dissected embryonic gut could be photographed in 80% glycerol on a depressed glass slide using a Zeiss Axioscope microscope. Fluorescence tagged antibody could be observed in the same way after careful dissection of the tissue to a suitable size. Sections, teased nerves and cultured cells were photographed using the Zeiss Axioscope with bright light, phase contrast, and fluorescent light settings. Kodak Tmax 100 negative films and Agfa professional 25 were used for bright field and phase contrast settings, and Ilford HP5 400 negative films were used for fluorescence photography. The films were processed in the our lab with appropriate developer and fixer and the final prints processed using an enlarger and an automatic dry print processor. For Nomarsky optics, tissues were crysectioned at 20uM and photographed using a Sony microscope (courtesy of Dr. Jon Clark) using Kodak Ektachrome 64 Tungsten positive films. Some of the fluorescence photography was taken using the departmental confocal system (Leica).

107 Po-1 Transcription Vector Shaded area-Coding Region

cc Sp6

MGS 1857 1031 904 608 306

► Antisense 3 - # - S ense ORF

Antisense Transcript ■►Cla IlSSOnt

Sense Transcript X ballO SInt

Figure 2-1 cDNA encoding the entire 1.8 kb Po coding sequence, Po-1 Table 2-1 cDNA clones used antisense sense gene clone vector RE site polymerase RE site polymerase source

BMP-4 PCR fragment PCR2 EcoRI 17 _* - c-ret pmcret? (2 .8 kb) pBluescript ( SK) Notl 17 Asp713 T3 V. Pachnis dll - 1 mouse dll - 1 pBluescript ( SK) Notl 13 - - D.Ish-Horowitz

DM-20 747bp coding sequence pBluescript (KS) BamHI 13 - - B. Zalc ErbB3 human ERbB3 bpl250-3’UTR pBluescript BamHI 17 Clal/Xhol T3 C. Birchmeier

FGF-3(int-2) mouse FGF3 with 5’UT pGEM3 Hindlll T7 -- C. Dickenson

Krox-20 750 bp Pstl-Apal fragment pBluescript (KS) BamHI 13 - - D. Wilkinson

PO rat PO-1 pGEM Clal 17 X bal SP6 G. Lemke/I. Griffith

SCIP 500 bp Dral 3’UTR fragment pSP71 Xhol SP 6 -- D. Meijer

Sek-1 1.5 kb EcoRI fragment 3’UT pBluescript (KS) Hindlll 17 - - D. Wilkinson serrate mouse Serrate/Jagged 1.8 kb T Rvin pKs EcoRI 13 Kpn T7 J. Lewise VVMNVVWWVWVkVVkSVVV«SWIrtr«%%'»SVKVVVVVVVVVV%SVV%VVVVVkVVVVVWWkV«VVVkVb*k*kivk%vvvvw ■WW%Vk*»NWWWW»W»VW%V%iVWW*%*i(V»V

: no sense probe was transcribed and a blank hybridization was used as a negative control. Chapter 3

The Differential Regulation of the Pq gene in the Two Mature Phenotypes of the Schwann Cell Lineage

This chapter describes data which form the basis for the major section of a published paper (Lee et al., 1997).

110 3.0 Table of contents 111

3.1 Introduction 112 3.2 Results 115 3.2.1 The Po gene is expressed at basal levels throughout embryonic development of the Schwann cell lineage 115 3.2.2 Axonal signals that inhibit Po expression 116 3.2.3 When myelinating cells are removed from axons, high Po expression falls to basal levels in a process that does not depend on active Po suppression 117

3.3 Discussion 119 3.4 Ilustrative materials Figure3-1 Po mRNA in the neural crest and in developing sciatic nerves 121 Figure3-2 Axon-dependent and reversible down-regulation of Po mRNA in adult non-myelining Schwann cells 123 Figure3-3 Removal of Schwann cells from axons leads to a fall in myelin related Po expression, while basal levels are relatively unaffected 124 Figure3-4 Schematic representation of Po mRNA distribution in cells of the Schwann cell lineage 125

111 3.1 Introduction The formation of a myelin sheath by Schwann cells is a particularly striking example of an inductive cell-cell interaction in which one cell, in this case the neuron, reversibly alters the phenotype of its neighbour, the Schwann cell (Bray et al., 1981; Mirsky and lessen, 1996). The major protein of PNS myelin, Po, comprises about 50% of the total myelin protein and, acting via homophilic interactions, has a major role in stabilising the myelin sheath (Lemke and Axel, 1985; D ’Urso et al., 1990; Filbin et al., 1990; Giese et al., 1992). Myelinating cells gradually deposit very large amounts of Po in the myelin sheath, and the relative abundance of Po mRNA rises about 30-40 fold from birth to its peak in actively myelinating nerves, while it falls to lower, stable levels in adult nerves (Lemke and Axel, 1985; Trapp et al., 1988; Stahl et al., 1990). Clearly, elucidation of the mechanisms that regulate Po gene expression is an important step towards understanding the cellular signalling that directs myelination.

Historically, three observations have had major impact in this area. Firstly, isolation of Schwann cells from axons prior to myelination prevents the striking increase in Po expression seen when Schwann cells myelinate, suggesting that expression of myelin proteins is induced by positive axon-associated signals and does not represent a default state (Mirsky et al., 1980). Secondly, removal of myelinating cells in developing or adult nerves from contact with axons by nerve transection results in a rapid reduction of Po expression, indicating that axonal induction of the Po gene remains reversible and that the presence of these signals is therefore required long term (Trapp et al., 1988). Thirdly, Po gene expression in Schwann cells has not been previously detected significantly before myelination starts in rodents, nor has Po been found in postnatal non-myelinating cells, suggesting that Po expression during myelination represents new gene expression specific to myelinating cells (e.g. Baron

et al., 1994).

It is timely to re-evaluate this picture of Po regulation, to take account of recent work, such as the finding that Po expression can be negatively regulated (for refs see below) and also to incorporate important but relatively neglected older observations. Thus, it

112 has been known for some time that although Po levels fall sharply in transected adult nerves, a low but significant amount of Po mRNA and protein remains (Poduslo et al., 1985; LeBlanc and Poduslo, 1990). This points to two modes of regulation, an extrinsically signalled, high expression superimposed on an intrinsically controlled constitutive baseline, although this matter has not been further systematically investigated. Intriguingly, Po is expressed in the chick neural crest and Po mRNA has been detected by the polymerase chain reaction (PCR) in whole rat embryos at embryo day 14 (E l4) (Bhattacharyya et al., 1991; Zhang et al., 1995). This leaves open the fundamental question of when and in which cells Po gene expression starts in mammalian glial cell development, particularly in light of recent findings that peripheral myelin proteins or alternatively spliced forms of them can be found outside the glial lineage (Pribyl et al., 1993; Parmantier et al., 1995). Lastly, there is evidence that Po gene expression can be suppressed as well as induced by cell-extrinsic signals. The first factor shown to have this effect was serum which caused a dose-dependent suppression of cAMP induced Po protein expression (Morgan et al., 1991) and similar effects have since been seen with fibroblast growth factor-2 (FGF2) (Morgan et al.,

1994), transforming growth factor- 8 (TGF8 ) (Mews and Meyer, 1993; Morgan et al., 1994) and neuregulin (Cheng and Mudge, 1996). Most strikingly, in co-cultures of neurons and Schwann cells, TGF 8 suppresses high Po expression and blocks myelination of axons, but allows formation of the normal mature relationship between unmyelinated axons and non-myelinating Schwann cells (Einheber et al., 1995; Guénard et al., 1995). Together these observations open the possibility that axonal suppression of Po expression is a part of the developmental repertoire. There has, however, been no description of such inhibitory effects of axons in normal Schwann cell development. The most radical interpretation of these studies of Po inhibition has been proposed by Cheng and Mudge (1996) who question the idea that the sharply elevated Po expression commencing around birth in actively myelinating cells is driven by positive axonal signals, and suggest instead that myelination-related levels of Po expression represent the constitutive state which, prior to myelination, is suppressed by inhibitory signals.

113 In this chapter I have examined most of the issues raised above. In contrast to previous observations on rodents, I find Po mRNA in a population of migrating neural crest cells, and show that it is also present in Schwann cell precursors from E l4 nerves and in embryonic Schwann cells prior to myelination, provided the assay is adjusted to higher sensitivity than that needed to detect Po in myelinating cells in vivo. This expression in embryonic Schwann cells is irrespective of whether the cells will subsequently myelinate or develop along the non-myelin pathway. Myelin- independent Po expression can also be detected at the protein level raising the possibility that Po has a role in cell-cell interactions in early glial development. Postnatally, the relative abundance of Po mRNA and protein is sharply elevated in the cells that myelinate, while cells not engaged in myelination continue to express basal levels of Po mRNA and protein initially, although Po mRNA subsequently falls to undetectable levels in mature non-myelinating cells, indicating the existence of suppressive signals in vivo. We show that the basal Po expression is constitutive, in the sense that it is independent of axons in vivo and continues under basal conditions in neuron-free cultures in the absence of specific Po promoting signals such as cAMP elevation. In contrast, we confirm that the high Po mRNA and protein levels in myelinating cells are truly reversible and appear to depend on positive axonal signals, by showing for the first time that myelin-related Po levels fall rapidly to basal levels when the cells are removed from axons, even under conditions that exclude simultaneous exposure to Po suppressing signals, e.g. serum in vitro or putative endoneurial inhibitory factors, such as TGFB secreted by macrophages, in vivo. Thus, the postnatal diversification of immature Schwann cells to form myelinating and non-myelinating cells involves axon-dependent amplification and suppression, respectively, of myelin-independent Po expression. In the Schwann cell lineage, this basal expression is a constitutive early phenotype that is likely to appear as one of the first signals of glial lineage choice in neural crest development.

114 3.2 Results 3.2.1 The Po gene is expressed at basal levels throughout embryonic development of the Schwann cell lineage In situ experiments were carried out on whole embryos at E ll (Figure lA). This revealed streams of Po"'’ cells on either side of the neural tube, forming a repeated pattern with the location and appearance characteristic of migrating neural crest cells (Erickson, 1989). Some of the hybridized embryos were double immunolabelled with antibodies against the neuron specific isoform of tubulin, B3, to reveal developing axons (Figure 1B,C). Observations of sections from these embryos suggested that among the crest cells, Po mRNA was localized selectively to clusters of cells, distributed among a larger number of Po negative cells (Figure IB). This has been confirmed by comparing the Po expression to the ErbB3 signal, which marks most of the neural crest population (see chapter 4). The Po positive crest cells were preferentially located near axons that are starting to grow from the neural tube at this stage. (Figure 1C).

To examine Po mRNA expression in Schwann cell precursors, hybridization experiments were carried out on sections from E l4-15 embryos (Figure 1D,E,F). A strong signal was seen in dorsal and ventral roots, within the DRG and in other peripheral nerves, including those well within the developing limb. Comparison of labelling patterns in sections developed for different time periods or treated with varying probe-concentrations showed that the Po signal was stronger and appeared earlier in dorsal and ventral roots compared with more distal nerves, indicating a proximo-distal gradient in Po mRNA abundance (not shown). Po expression in embryonic Schwann cells prior to myelination was examined in sections of E17 embryos (not shown) and teased preparations of the sciatic nerve and sympathetic trunk of El 8 embryos (Figure 1G,H,I). An unambiguous Po hybridization signal was seen not only in pre-myelinating Schwann cells of the sciatic nerve but also in cells of the E l 8 sympathetic trunk although about 95% of the cells in this nerve remain non­ myelinating in the adult. To estimate the relative strength of the Po hybridization signal in Schwann cell precursors and myelinating Schwann cells, sections of PIO sciatic nerve and E14 embryos were hybridized using a range of dilutions of the Po

115 probe with all other experimental parameters constant. Probe concentrations required for clear visualization of Po mRNA in postnatal day 10 sciatic nerve sections were approximately 25 fold lower than those required to generate a strong signal in E14 nerves (not shown).

3.2.2 Axonal signals that inhibit Po expression Using detection-sensitivity similar to that used above to detect Po mRNA in embryonic nerves we now examined Po mRNA expression in adult nerves (Figure 2A-G). As expected, a very high Po signal was seen in myelinating cells, and the labelling intensity showed a positive correlation with the thickness of the myelin sheath in agreement with previous work (Griffiths et al., 1989). Surprisingly, however, no Po mRNA was detected with these methods in the mature non­ myelinating Schwann cells. These cells remained Po negative, both in the sciatic nerve and the cervical sympathetic trunk, while E l8 sciatic nerve and trunk processed identically gave a clear Po signal; similar conditions also resulted in a clear signal in nerves of EI4/I5 embryos. Therefore the basal Po gene expression seen in certain neural crest cells, Schwann cell precursors and embryonic Schwann cells, appears to be down-regulated in adult non-myelinating cells. To explore this further we carried out three types of nerve-transection experiments. Firstly, the adult sympathetic trunk, which is largely unmyelinated, was transected and the cells of the distal stump examined in teased preparations 9 days later (Figure 2H). Immunohistochemistry with MBP antibodies and examination by phase contrast optics confirmed the expected disappearance of the relatively few myelin sheaths seen in normal trunks. In situ experiments showed that this was accompanied by disappearance of any cells expressing very high levels of Po mRNA. Surprisingly, most of the cells in the distal stump now gave a clearly detectable low level Po mRNA signal, comparable in intensity to that seen in the cells of trunk and sciatic nerve in E l8 embryos (Figure 2E,F,G). Since about 95% of the Schwann cells in normal trunks are non­ myelinating and Po mRNA negative (above), this means that in non-myelinating cells, loss of axonal contact results in up-regulation of Po gene expression. Thus loss of axonal contact in vivo results in Po mRNA expression in myelinating and non­ myelinating cells moving from opposite directions towards a common basal level.

116 The constitutive nature of this basal Po expression in adult Schwann cells was further indicated by finding a similar low intensity hybridization signal in the cells of the distal stump of the sciatic nerve 5 weeks after nerve transection (Figure 21,J). We examined the regulation of Po protein in cultures of cells from adult cervical sympathetic trunk.

Since basal Po mRNA levels are low compared to that seen in myelinating cells, the same might hold for protein levels and the immunohistochemical assay was therefore adjusted to a higher sensitivity by applying high concentration of antibody. When this assay was applied to 3 h cultures, strong Po immunolabelling was seen in essentially all the Schwann cells from newborn and 4 day old nerves, but also in most or all E l4 precursors and E l8 Schwann cells (not shown, Lee et al., 1997). When cells of the adult cervical sympathetic trunk (CST) were plated in serum free defined medium without pretreatment that involved exposure to serum, substantial level of Po were detected using the sensitive method of detection for Po prtein within 5 hours (Figure 2K,L).

3.2.3 When myelinating cells are removed from axons, high Po expression falls to basal levels in a process that does not depend on active Po suppression We examined Po mRNA by in situ hybridization using a DIG labelled probe in cultures from newborn nerves that were plated and maintained for 7 days as described above (Figure 3A,B). A strong signal was obtained from 25-35% of the cells after 1 day in vitro, while the rest of the cells showed weak labelling corresponding to basal Po expression. As with high protein expression, the number of cells with a high mRNA signal fell radically during the culture period. The defined medium used in these experiments contained 5 p.g of insulin, sufficient to stimulate type 1 IGF receptors, or alternatively IGF-1, and progesterone, dexamethasone, triiodothyronine (T3) and thyroxine (T4). Although all of these components have previously been shown either to promote myelination directly or to enhance the synthesis of myelin proteins, including Po (Warringa et al., 1987; Barakat-Walter et al., 1992; Tosic et al., 1992; Koenig et al., 1995; Stewart et al., 1996), we nevertheless carried out control experiments from which these components were absent to further exclude any

117 inhibitory signalling that might affect Po expression. These experiments show that in myelinating Schwann cells the relative abundance of Po mRNA falls radically to basal levels when the cells are removed from contact with axons, even in the complete absence of any factors known to suppress Po expression. The basal levels, in contrast, continue to be expressed in the routine serum-free medium employed in these experiments and are, in this sense, constitutive.

118 3.3 Discussion We show here that in the rat, constitutive, basal expression of the Po gene can, using sensitive methods, be traced back as far as the neural crest. We found no evidence for Po expression outside cells of the peripheral glial lineage. Thus, Po is likely to represent one of the earliest markers of glial differentiation in both mammalian and avian neural crest (Barbu, 1990; Bhattacharyya et al., 1991, Zhang et al., 1995). Therefore the massive Po expression in myelinating cells represents an axon-induced amplification of pre-existing basal levels rather than new gene activation. We also show that signals that inhibit Po are not required for the sharp reduction in Po mRNA and protein expression that follows removal of myelinating Schwann cells from axons. Our experiments suggest, however that inhibitory signals have a role in the development of non-myelinating cells in vivo (Mews and Meyer, 1993).

By combining in vivo and in vitro observations it is now possible to distinguish three broad categories of Po expression (Figure 4). (I) High, induced levels in cells forming myelin. (II) Lower, but clearly detectable levels seen early in development in some neural crest cells, in Schwann cell precursors and early Schwann cells and in cultured cells; this basal level is constitutive in the sense that it is present in denervated Schwann cells in vivo and in Schwann cells cultured under minimal conditions without inhibitory signalling molecules. (Ill) Suppressed levels seen in mature non­ myelinating cells in vivo and in cells cultured with Po inhibitory signals such as serum, NDFB, TGFB or FGF. This model accords with previous findings. These include: the rapid rise in Po mRNA that occurs at the onset of myelination and its rapid fall on denervation (Lemke and Axel, 1985; Trapp et al., 1988; Stahl et al. 1990); the basal levels of Po and MBP that exist in adult Schwann cells deprived of axonal contact and the Po mRNA that persists in cultured neonatal cells (Poduslo et al., 1985; LeBlanc and Poduslo, 1990; Morgan et al., 1991; Morrison et al., 1991). It also accords with the finding that mouse Schwann cells continue to express low levels of Po protein in vitro in the absence of myelin assembly (Burroni et al., 1988) and the similar observation made more recently in the rat (Cheng and Mudge, 1996). The existence of significant Po expression in the Schwann cell lineage throughout embryonic development, its suppression in mature non-myelinating cells and re­

119 appearance on denervation indicates that Po might have a role in development and regeneration that is separate from its function in the myelin sheath (D'Urso et al., 1990; Filbin et al., 1990; Schneider-Schaulies et al., 1990). Our observations also suggest that the Po promoter might contain regions responsible for three distinct expression patterns; up-regulation, down-regulation and basal expression that is lineage specific and activated early in development.

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FIG. 3-1 Po mRNA in the neural crest and in developing sciatic ner\ es. (A) Whole-mount in situ hybridization shows clear labeling in the position of tlie migrating neural crest cells. Bar, 350 pm. (B and C) Double labeling for P q m RNA (B) and p S tubulin using monoclonal antibody TUJl to label neurons (C). Note that in these sections, Pp-positive neural crest cells are found in the same region as the early peripheral axons growing out from the neural tube (NT). Bar, 40 pm. (D, E, and F) Pp mRNA in Schwann cell precursors. (D) A transverse section through the spinal cord and surrounding tissues with clear hybridization signal in the dorsal and ventral roots of the embryo, in cells within the dorsal root ganglion, and in the developing sciatic nerves. In (E) Pp signal is present in nerves (arrow) within the developing hindlimb, as well as in the more proximal part of the sciatic nerve. Bar, 1000 pm. (F) shows a higher magnification view of a transverse section through two nerves within the developing limb. 20 to 30 and 30 to 40 Pp-positive Schwann cell precursors are distributed throughout each nerve, respectively. Bar, 40 pm. (G, H, and I) Teased preparations showing that in E18 sympathetic trunk and sciatic nerve basal levels of Pp mRNA are present in most Schwann cells. E18 sympathetic trunk treated with a sense probe to Pp is shown in (H). Bar, 50 pm. Figure 3-2 Axon-dependent and reversible down-regulation of Pq mRNA and protein in adult non-myelinating Schwann cells. (A,B,C,D) Normal adult sciatic nerve, teased and triple labelled. (A) Immunofluorescent labelling with antibodies to MBP to reveal myelinated fibres. (B) Phase contrast micrograph showing several myelinated fibres and an unmyelinated fibre (arrowed). (C) Immunofiuorescent labelling with antibodies to GAP-43 to reveal the unmyelinated fibre (nucleus of non-myelinating Schwann cell arrowed). (D) Bright field micrograph showing strongPq mRNA labelling in the perinuclear area of three myelinating Schwann cells. Note that the non-myelinating Schwann cell (area of nucleus arrowed) is unlabelled, showing down-regulation of Pq mRNA. Bar, 15 pM. (E,F,G) Normal adult sympathetic trunk, teased and double labelled. (E) Immunofiuorescent labelling with antibodies to MBP shows two myelinated fibres; the nucleus of one of the two myelinating cells associated with the upper MBP positive fibre is double arrowed. (F) Phase contrast view of the same field, revealing many non-myelinating cells (nucleus of one of the cells is arrowed), in addition to the two myelinated fibres (double arrow indicates the same cell as in (E)). (G) Bright field micrograph showing Pq mRNA labelling in the perinuclear area of three myelinating Schwann cells but a complete lack of labelling in the non-myelinating cells, indicating down-regulation (double and single arrows indicate the same cells as in (F)). Bar, 15 pM. (H ) Adult sympathetic trunk, 9 days after transection, teased. Pq mRNA is clearly visible in most cells in this low power micrograph, demonstrating that up-regulation to basal levels has occurred in this time. An unlabelled blood vessel is arrowed. Bar 25 pM. (I,J) Adult sciatic nerve, teased distal stump, 5 weeks after transection. Note that the levels of Pq mRNA are similar to those seen in the transected sympathetic trunk. (I) Bar as in (H). (J) A higher power view showing the nuclear regions (one indicated by a bracket) of a few fibres. Bar, 7.5 pM. (K,L) PO protein detected in 5 hour after plating in cells from adult cervical sympathetic trunk. Arrow and arrowhead point to an originally non-myelinating Schwann cell and a fibroblast, respectively. Note that the level of flourecence in the originally non-myelinating Schwann cell is higher than in a fibroblast (L). Bar, 40 pM.

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Figure 3-3 Removal of Schwann cells from axons leads to a fall in myelin-related Pq expression, while basal levels are relatively unaffected. Bright field micrographs showing Pq revealed by in situ hybridization in newborn Schwann cells cultured in defined medium for 1 day and 7 days. In the 1 day culture (A) those cells about to myelinate show much higher levels of Pq than others, whereas by 7 days (B) these

heavily labelled cells are no longer visible and all cells contain basal P q levels. In control experiments in which insulin was omitted with or without the addition of progesterone, T3 and T4 and dexamethsone or the insulin level was lowered to 5 ng/ml with or without progesterone, T3 and T4 and dexamethasone the drop in Pq levels was not significantly altered compared with results obtained using medium containing either high insulin or alternatively 100 ng/ml IGF-1, progesterone, T3 and T4, and dexamethasone. Bar, 24 pM. P q in Embryonic and Postnatal Schwann Cells

Myelin Schwann cells

Neural Precursors/ Non-myelln Schwann crest early Schwann celts-no Schwann cells axonal cells contact

FIG. 3-4 Schematic representation of Pq mRNA distribution in cells of the Schwann cell lineage. Shading within the ellipses gives a rough estimate of relative mRNA levels in key cells at different stages of development and after nerve transection. Chapter 4

The Expression of Pq Gene in Neural Crest Cells and the Generation of Schwann Cell Precursors From the Neural Crest

126 4.0 Table of contents 127 4.1 Introduction 129 4.2 Results 135 4.2.1 Analysis of Po mRNA at early developmental stages of the rat using the RT-PCR method 135 4.2.2 Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization at the trunk level 136 4.2.3 Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization at the head level 139 4.2.4 Po expression in the DRG and trunk spinal nerves at the Schwann cell precursor stage at E14 and in Schwann cells at E l7 139 4. .2.5 Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization in the gut 140 4.2.5.1 Development of the gut 140 4.2.5.2 Po expression in the gut; comparison to c-ret 141 4.2.6 Po and type 3 ptubulin are expressed in the neural crest in vitro 143 4.3 Discussion 145 4.3.1 Po is expressed in the migrating neural crest, but not in the newly emerging population. 145 4.3.2 Po is expressed in a sub-population of the migrating crest which does not include the melanocyte lineage 147 4.3.3 Po positive cells in the ventral exit zone may not derive exclusively from the migrating neural crest 148 4.3.4. The spatial distribution of Po strongly suggests it is expressed in immature peripheral glial cells 149 4.3.5 Po is expressed in the glial lineage after the separation of neuron and glial fates 150 4.3.6 Po is the known earliest gene to be expressed in the PNS glial lineage 151 4.3.7 Po is expressed in the migrating neural crest in the gut at the same time as the neurofilament positive vagus nerves innervate

127 the gut, and one day after the first appearance of c-ret positive neuroblasts. 154 4.3.8 The transient expression of Po during embryonic development could be universal to PNS glia of different terminal phenotypes 155 4.4 Ilustrated materials Figure 4-1 RT-PCR of Po in developing embryo 158 Figure 4-2 Po expression in ElO 160 Figure 4-3 Po expression in El 1 : comparison to TUJ-1 160 Figure 4-4 Po expression in E12: comparison to TUJ-1, ErbB3 and Krox-20 160 Figure 4-5 Po expression in E l4 161 Figure 4-6 Developing enteric nervous system 162 Figure 4-7 Neural crest culture 164 Figure 4-8 The periodic pattern of Po expression in the migrating neural crest 165 Figure 4-9 A summary of Po expression during the generation of peripheral glial phenotypes 167

128 4.1 Introduction In the last 20 years studies of the neural crest have generated a large body of facts and models about the development of this elusive structure. The main but somewhat contradictory observations about the neural crest are its heterogeneity and multipotentiality. On the one hand clonal study of single cells reveals the plasticity of migratory neural crest cells, by showing their ability to generate a vast variety of different derivatives depending on the extrinsic cues they are exposed to. On the other hand reports on the existence of fate-restricted precursor cells argue against the model that neural crest cells are uncommitted during migration. Elegant back-transplantation experiments by Ayer-Le Lievre and Le Douarin (1982) and Fontaine-Perus et al. (1988) showed that even in the DRG, where the derivatives of neural crest differentiated to the terminal phenotypes, some glial cells were able to give rise to neuronal cells when back transplanted to the ventral pathway. The isolation of self- renewing stem cells for neuronal and glial cells directly showed the existence of uncommitted, hipotential cells (Stemple and Anderson, 1992). These data support a mechanism in which in situ factors induce the specification of the correct phenotyes. At the same time, cells committed to specific lineages were found in the migratory neural crest population (Bronner-Fraser and Fraser, 1989), and some sub-populations of neural crest migrate to their precise position only when their fates are already specified (Erickson and Goins, 1995). These data support the idea that the appearance of differentiated cells in precise locations is the result of the differential localization of developmentally restricted precursors.

To account for the co-existence of multipotent stem cells, precursors that generate a limited range of derivatives, and fate restricted cells in the migrating neural crest population, one could propose a model in which the fate of some multipotent neural crest was restricted during migration, while some remain multipotent and capable of generating cells of diverse phenotypes when properly stimulated. Like all questions to be answered about embryonic development, all studies concerning this pluripotent, transient structure can be boiled down to be three questions: where, when, and what. Indeed all the phenomena in embryonic development are, almost without exception, an interplay of the location, the timing and the phenotypes. For every piece of

129 information to be jigsawed into this picture, precise timing and location of the differentiation events is very much needed. However, no matter how carefully conducted, the usefulness of research to date on the neural crest has been hindered by the lack of reference to the exact time point and location of the differentiation events. There is no doubt of the importance of these experiments in confirming the multipotentiality and heterogeneity of neural crest, but, because of the nature of the approaches used, the outcome of individual studies depends on the successful sampling of the diverse and mobile structure of the neural crest (see also Henion and Weston, 1997). For example, in most of the single cell culture experiments, the cells were either secondary clones from cells derived from primary neural tube outgrowth populations (Sieber-Blum and Cohen, 1980; Stemple and Anderson, 1992), or obtained by dissociation of mesencephalic neural crest cells already in their migrating pathway (Baroffio et al., 1988, 1991; Dupin et al, 1990). The neural crest cells certainly face selective survival or proliferate in the primary outgrowth in the ‘full differentiating medium’, which contains serum and embryo extract. Cells which have emigrated from the neural tube certainly would also have been influenced by the mechanical force generated by the emigration and certain environmental factors at the time of the dissection.

Ideally a repertoire of markers for each developmental stage would provide a useful tool to map the stages of restriction of the lineage. Surprisingly there are very few, or even no markers for the stage when individual precursors segregate toward a specific fate. The use of markers is clearly demonstrated by research on the 0-2A lineage (Raff, 1989, 1992; Raff et al, 1983, 1985, 1988; for review see Richardson et al., 1990) and on the development of the Schwann cells (for review see Mirsky and lessen, 1990, 1996; lessen and Mirsky, 1992, 1993). Antigens characterized in these in vitro systems probably change less rapidly than in the neural crest cells, where cells are differentiating in a very short time period. In the rat, the neural crest in the trunk area starts to migrate around E9-E10 (Erickson et al., 1989). At E ll the neurons in the trigeminal ganglia already express early neuronal antigens (Stainier et al., 1990, 1991). At E12 at the thoracic level, neurons in the dorsal and ventral roots and DRG are clearly visualized by the presence of neurofilament, recognized by M0-3D and

130 MO-3 A antibodies (Chen and Chiu, 1992). At ElO in the mouse (developmentally equal to E l2 rat) immunoreactivity, visualized by the antibody TUJ-1, to the (33 form of tubulin, an early neuronal marker, is prominent in the cranial ganglion and nerves. The cranial nerves are already reaching target sites, with cells identified by their ErbB-3 gene expression and supposedly of glial lineage, distributed along the nerves (Meyer and Birchmeier, 1995). This demonstrates that in 1-3 days the premigratory neuroepithelium cells migrate to their target site and advanced differentiation is well underway. Unfortunately many of the markers of neural crest or early markers which claim to be specific for fate-restricted lineages have not been characterized in a well- defined developmental time frame, when the work was done in vivo. For example, some markers claimed to be expressed in the neural crest proved to be specific to immature neurons (Lo et al., 1994; Lo and Anderson, 1995). The rostral-caudal difference in developmental stage could be as much as one day difference in development at the same rostral-caudal level (Serbedzija et al., 1990). Moreover, the exact rostral-caudal level of the sections studied was not always reported, which sometimes causes confusion as to the extent of differentiation of the cells which express the marker. As for the in vitro studies, most lineage markers have been characterized in culture one week after the neural crest outgrowth or even in secondary neural crest clones cultured over a period of 2 weeks (Sieber-Blum and Cohen, 1980; Baroffio et al., 1988, 1991; Dulac et al., 1988; Smith-Thomas and Fawcett, 1989; Dupin et al, 1990; Stemple and Anderson, 1992).

A detailed characterization of the exact time point and the expression pattern of molecular phenotypes of the neural crest derivatives is much needed, to be able to monitor directly the restriction of cell fates during the differentiation of neural crest. My study of the Po gene in the Schwann cell lineage has suggested that the major myelin protein Po is expressed constitutively in the glial lineage (Lee et al, 1997). Using sensitive methods, it was found that the Po gene, a classical myelination gene, is expressed as early as the mid gestation stage in the rat. This finding is different from the traditional view of Po being always associated with myelination. This, plus the conclusion that Po gene is constitutively expressed in embryonic Schwann cells and in mature Schwann cells, when inhibitory signals are absent, points to a

131 conclusion that Po maybe a marker for the glial lineage in the neural crest. Because Po levels are regulated by axons or equivalent extracellular factors, I hesitate to use the word ‘marker’ for the Po gene. Ideally a good marker should not be significantly regulated at the basal level by extrinsic factors. Instead, I use the phrase ‘early phenotype’. This indicates that a gene is an early trait of a cell lineage and to some extent could serve as a good marker.

In common with all molecules used as cell type specific markers for the first time, the problem is the absence of reputable markers against which the marker can be compared. As a result, establishing a first marker involves a lot of morphological studies. My first approach was to examine the expression pattern of Po temporally in the embryos, and correlate it spatially and temporally with the known pattern of neural crest derivatives of known developmental potential. In an effort to be true to the timing and the environmental (or location) influence on the gene studied, many studies were done on the gene in situ. Embryos were fixed as whole mounts and sectioned to analyze Po expression in the real migrating population of neural crest. With the precise location of Po gene expression in the embryos, the degree of heterogeneity and fate restriction within the migrating neural crest is directly demonstrated. The spatial distribution of Po also strongly suggests that it is expressed in the glial lineage. In vitro study of the neural crest gives confirmation on the extent of fate restriction of Po expressing cells.

Several antibodies, originally were directed against cell type specific antigenic markers of neural crest derivatives in adulthood, were expressed in embryos as well. In the glial lineage several molecules of this nature were reported to be expressed in the second half of gestation. These include SI00, low affinity NGF receptor, Gal-C, PLP, MBP, PMP-22, SCIP and Krox-20 (references see 1.2.2-5). The expression of these molecules marks different developmental stages of the peripheral glial lineage. SI00 is conventionally used as a glial marker, and the presence of this molecule in the cytoplasm marks the transition from Schwann cell precursors to Schwann cells (lessen et al., 1994). Low affinity NGF-R, recognized by monoclonal antibody 192 IgG, has been used to select the neural crest population in the caudal segment

132 outgrowth (Stemple and Anderson, 1992). Gal-C, 04 antigen, PLP, MBP, and PMP- 22 are structural lipids and proteins of myelin. Gal-C and the antigen recognized by 04 have been reported to be expressed before the onset of myelination in peripheral glia (lessen et al., 1985; Mirsky et al., 1990). PLP, MBP and PMP-22 were reported to be expressed well before the onset of myelination in the CNS but not in the PNS (Ikenaka et al., 1992; Timsit et al., 1992, 1995; Nakajima et al., 1993; Parmantier et al., 1995). ErbB3 is one of the major receptors for neuregulin and it is expressed by developing Schwann cells and their precursors (Meyer and Birchmeier, 1995). SCIP and Krox-20 have been shown to be necessary for myelination and both are expressed during embryogenesis (Topiko et al., 1994; Murphy et al., 1996; Bermingham et al., 1996; Jaegle et al., 1996). The above mentioned molecules are expressed in a well- timed sequence in the second half of gestation, as Schwann cell precursors derived from the migrating neural crest differentiate into Schwann cells. In this chapter the precise timing and pathways of expression of Po are documented and a comparison of Po to known early phenotypes expressed in the Schwann cell lineage is presented.

I was also interested in comparing Po expression to markers of the early neuronal lineage. Two known early markers of the neuronal lineage are neurofilament and tubulin (Chen and Chiu, 1992; Memberg and Hall, 1994). The low molecular weight neurofilament (NF-L, 68kD) first appears in the cervical level at E l2 in rat (Chen and Chiu, 1992), which is the same time as the medium molecular weight neurofilament (NE-M, 160 kD). The expression of P3 tubulin in immature neurons is earlier than NF-M (Memberg and Hall, 1994). A study of neuron-specific P tubulin, detected by the monoclonal antibody TUJ-1, showed that expression of neuronal traits, i.e., restriction to the neuronal fate, is earlier than the final mitosis. In other words, cells can enter the neuronal lineage while still proliferating (Memberg and Hall, 1994). To date no neuronal marker marking an early stage after the lineage segregation of glia and neurons has been described in rat. In other words, TUJ-1 immunoreactive cells which have proliferating and rounded cell bodies are the earliest phenotype identified in the neuronal lineage of rat. In my study attempts were made to examine the expression pattern of the TUJ-1 immunoreactivity and Po gene expression in the neural crest outgrowth system in tissue culture. .

133 Another problem with the identification of specific lineage markers is that some molecules described may as such in fact not be so. Some antigens have been described as marking the early migrating neural crest or early neuronal precursors on the basis of a broad correlation with an exact developmental stage or specific cell population, but their specificity is far from clear. For example, CRABP (cellular retino acid binding protein) was suggested to be a marker for the neural crest. The timing of expression is correct, but the expression pattern is far too broad for it to be restricted solely to the neural crest (MJ Lee, unpublished data). This is the same for the low affinity NGF-receptor, recognized by monoclonal antibody 192-IgG. It has been shown to be expressed at the target site of nerves, i.e., the skin and the muscle, in addition to the neural crest (Yan and Johnson, 1988). These type of molecules still serve as good markers in culture when non-neural cells can be distinguished or excluded by other methods.

The Schwann cell precursor is a transitional stage in the Schwann cell lineage which has been characterized in the sciatic nerve of E14 and 15 rat embryos. (lessen et al., 1994; Dong et al, 1995; Gavrilovic et al., 1995). An objective of my study was also to study the transition of neural crest cells to Schwann cell precursors. The difference between Schwann cell precursors and Schwann cells is well documented: the precursors are S100 negative and need neuronal factors to survive, while the Schwann cells are SI00 positive, and can survive in the absence of neuronal factors under conditions where precursors die (lessen et al., 1994; Gavrilovic et al., 1995). Neuregulin alone is able to support the survival of these precursors and promote the transition of precursors to Schwann cells without proliferation (Dong et al, 1995). The Schwann cell precursors are different from neural crest as well: neural crest cells in culture are GAP-43 negative under conditions where precursors are positive, and the mobility of the two cell types is distinctly different. However, the transition from neural crest cells to Schwann cell precursors has been comparatively less studied because of a lack of suitable markers. We hoped that a detailed study of Po during early ontogeny might shed light on this subject.

134 4.2 Results 4.2.1 Analysis of Po mRNA at early developmental stages of the rat using the RT-PCR method To test whether Po mRNA is expressed during early embryogenesis in the rat, total RNA was extracted from El 1 whole embryos, and compared to RNA extracted from sciatic nerves of E14 and P4. RT-PCR detection of Po mRNA in rat has been reported by other groups (Brunden et al., 1992; Zhang et al., 1995). In this study oligonucleotide primer pairs complementary to exon 4 and 6 were used, following the method of Brunden et al (1992). Since whole embryos of E ll were used while only sciatic nerves were used for E14 and P4, the intensity of the amplified band cannot be used as an indication of the relative abundance of Po mRNA per cell at these ages, therefore no attempt was made to adjust the amount of RNA for reverse transcription to equal the same amount from the same number of cells. In this circumstance, an equal amount of cDNA, which is transcribed from an equal amount (500 ng) of RNA, was used for amplification. A band that has a similar size to the expected 181 bp segment, corresponding to a region spanning exons 4 through 6 of the Po transcript, was present (Figure 4-1). An unexpected band of approximately 600 bp came up when the annealing temperature of PGR was set at 52^C, but disappeared when the annealing temperature was set at 60^ C (not shown). A possible source of this larger band is the genomic DNA, which, if amplified, would give a DNA fragment of 427 bp calculating the two introns between exon 4 and 6. However, since the band of higher molecular weight disappeared after raising the annealing temperature, this band is probably due to non-specific binding of the primer.

Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization To precisely locate Po mRNA in the embryos, in situ hybridization using digoxygenin-labelled riboprobe was carried out on whole embryos or sections of embryos. The use of digoxygenin has the advantage over radiolabelled probe in that it is possible to achieve cellular resolution. It should be noted that the cell types and anatomy of the neural crest derivatives in head area and in trunk area are significantly different. Furthermore, the formation of the enteric nervous system involves

135 migration of neural crest from the cranial area to the gut region. Therefore my description of Po expression and discussion is based on the division of the body into trunk, head, and the gut regions.

4.2.2 Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization at the trunk level Whole mount embryos at ElO, 11 and 12 were chosen to analyze the spatial correlation of the expression of the Po gene and the migrating crest population The blue/purple color precipitation of the Po ISH in the whole mount embryos was seen in the ElO rat embryo in the cranial area (Figure 4-2). Ell, 12 embryos were hybridized with Po probe and immunostained with TUJ-1 antibody, which stains growing neurites and immature sensory neurons (Memberg and Hall, 1994) (Figure 4- 3 A,B and Figure 4-4A,B). The Po signal was visualized by a mixture of NBT/X-P solution, and the TUJ-1 was visualized by incubation in DAB solution, as described in chapter 2.

At Ell there is an active spectrum of migrating neural crest ranging from premigratory stage to mid migration stages (Erickson et al, 1989; Serbedzija et al., 1990). The blue/purple color precipitation of the Po ISH in the whole mount embryos was seen as repeating wedged or squared streams of cells, spreading from the edge of the tube (Figure 4-3A). Each stream was contained within one somite. In the more caudal somites, the Po positivity was seen as slim stripes running dorsal-ventrally 1-2 mm lateral from the tube midline. These cells were not restricted to the anterior half of the somite when they first emerged from the tube, but soon after they migrated out of the near vicinity of the dorsal midline of the neural tube, the positive cells were seen to be arranged in 2 to 3 finer streams, and the migration was restricted to the anterior half of the somite. In the more rostral, and hence more mature somites, the positive cells formed a wedge, with the exception of the first 3 somites, due to the lack of neural crest derivatives in this area. The ventral most, thus supposedly front of the migrating crest was always restricted to the anterior part of the somite (Figure 4- 3 A, also Figure 4-4C), while nearer to the dorsal edge of the neural tube, the positive cells spanned nearly the whole length of the somite and formed the fat end of the

136 wedge. This is typical of neural crest populations in that the restriction to the anterior part of the somite only happens after the cells migrate some short but significant length ventrally. This is often less addressed than the phenomenon of restriction to the anterior somite, as it is not always seen in classical longitudinal sections.

A longitudinal line of dark ISH precipitation was seen running across two thirds of the length from the top of the wedged form beginning from the first somite and fading away at around the 10th to 12th somite (Figure 4-3A, arrow). Results from segments partially digested with collagenase to expose the somites showed that this line consisted of cells situated at the area where the motor axons first meet the migrating crest cells. At the 3rd to 6th somites, the blue/purple color was also seen much more ventrally in the area where future sympathetic ganglia would locate. The level of Po expression went down gradually towards the tail of the embryo. There were no Po signals in the last 5 to 6 somites.

The migration of the neural crest continues on the next day (El2). Neural crest migration into the periphery is initiated in an anterior to posterior wave (Erickson et al, 1989). The anterior to posterior progression is most obvious when forelimb and himdlimb levels are compared in the same embryos. The correlation of Po expression to the migrating crest population was studied with serial cross sections of the embryo. The ventral extent of Po expression forms a repetitive pattern, repeating itself every 200 p M on average, which is the length of a somite. This pattern is also seen in a longitudinal section of E l2 embryo hybridized with Po probe (Figure 4-4C), where the Po signal of wedged blue precipitation was seen only in the anterior half of the somite, and not in the posterior. In cross sections at the hind limb level Po signal was present in a location typically described as the ventral-lateral pathway of neural crest, as shown in the cross section of the anterior half of the somite (Figure 4-4F). Note that the Po is not present in cells of the migration pathway as soon as they emerged from the neural tube, and it is not expressed in the posterior half of the somite (Figure 4-4G), apart from two points which are probably the ventral exit zone for motor axons and in the notochord. A cross section of the whole mount embryo confirmed that these Po signals in the ventral exit zone run parallel to the neural tube to form a

137 longitudinal line which is only interrupted between somites. Sections from the same rostral-caudal level of same age of embryo hybridized with Krox-20 were compared. Po was expressed at the ventral exit zone when it is still Krox-20 negative(Figure 4- 4J). This showed that the Po is expressed earlier than Krox-20 at this location. However, cells in the entry/exit zone of the cranial nerves of the same embryo were already Krox-20 positive (Figure 4-4K), indicating the temporal proximity of Po and Krox-20 expression.

In the front limb level the derivatives of neural crest have aggregated to form the DRG, as shown by the presence of TUJ-1 antigen. The Po signal was present in the now substantial structure of the DRG, as well as along the length of the spinal nerves (Figure 4-4H, I). The crest-derived cells that coalesce to form the young DRG were observed as early as E12, as shown by the presence of TUJ-1 antigen (Figure 4-4B). At this stage the Po positive cells were seen in the entire area of these condensations (Figure 4-1). At the later stage of E14 the Po signal was down-regulated in the DRG itself and was only seen in the roots and spinal nerves (Figure 4-5), and this was similar in the mature adult DRG. At a more rostral level the Po was seen in the anlage of the sympathetic chain (Figure 4-4A).

E l2 rat embryos were hybridized with ErbB3 probe and cryosectioned as described for Po. Cross sections of the hind limb level were compared to those hybridized with Po probe, and sections chosen for comparison were from similar anterior-posterior positions within the somite. This was done by counting a similar number of sections from the boundary of the somite. The ErbB3 signal was very similar to the Po signal, with small but significant differences. In the anterior half of the somite the ErbB3 was seen in the cells between the ectoderm and the neural tube and in the wedge between the neural tube, dermamyotome, and the overlying ectoderm (Figure 4-4D). These bands of expression on the two sides of the neural tube were, or were nearly, continuous with the top of the neural tube. This continuity stretched ventrally, first between the dermatome and neural tube and, as the cells migrate more ventrally, near to the neural tube. It then extends laterally when reaching the exit point of the motor axons (Figure 4-4D). In the posterior half of the somite, the ErbB3 was expressed in

138 a group of aggregated cells sitting in a wedge between the neural tube and dermamyotome but above the sclerotome. The location of these grouped cells was nearer to the dermatome than to the neural tube, and the expression was highest on the ventral extreme (Figure 4-4E). Some cells in the myotome were ErbB3 positive, in agreement with results reported by Meyer and Birchmeier in mouse (1995). However, this signal in myotome is only significant in the anterior half of the somite (Figure 4-4D)

4.2.3 Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization at the head level The Po signal was first detected in the head of ElO rat embryo. The Po positive structures include the cephalic crest and the otic placode/cup (Figure 4-2). The next day at E ll the cephalic crest was seen to condense to form the trigeminal ganglion (Figure 4-3 A). At this stage, the otic placode/cup has already formed the otic cup and it was very Po positive (Figure 4-3A). At E l2 all the cranial ganglia and the cranial nerves were formed and this was clearly demonstrated by TUJ-1 staining (Figure 4- 4B). All the cranial ganglia were Po positive (Figure 4-4A). These include the trigeminal (V), acoustic-facial(VII/VIII), glossopharyngeal (also petrosal) (IX), nodose (X), accessory (XI), hypoglossal(XII) (Roman numbers in parenthesis indicate the numbers of the cranial nerves). The cranial nerves were positive as well. These include oculomotor (III), ophthalmic and maxillary (V) acoustic nerves (VII), facial nerves (VIII), and the nerves associated with glossopharyngeal (also petrosal) (IX), nodose (X), accessory (XI), hypoglossal (XII) ganglia. Olfactory nerves were positive later on at E l3 (see chapter 5 Figure 5-8A), and the optic nerves, of which the glial cells are of CNS origin, were negative. The rest of the cranial nerves (VI, abducens VI) were too small to be examined. The Po expression in placode derived structures is detailed in chapter 5.

4.2.4 Po expression in the DRG and trunk spinal nerves at the Schwann ceil precursor stage at E14 and in Schwann cells at E17 With ISH on sections containing almost the entire length of the sciatic nerves using Po riboprobe, the Po mRNA signal was shown by blue/purple precipitation as before.

139 The nerves which were undergoing active growth were visualized with fluorescein by post ISH staining using a polyclonal GAP-43 antibody and anti-rabbit Ig fluorescein. In E14 various levels of Po mRNA are expressed in regions of the PNS including the DRG, dorsal roots, ventral roots, and sciatic nerves. The blue-purple signal in the dorsal and ventral roots is very dark, and signal was absent in the CNS (Figure 4-5). The signal in the DRG was of very low level and sporadic. The finger-like penetration of the precursor Schwann cell cytoplasm which was observed in electronmicroscopy (lessen et al., 1994) was not seen in this 7 |im microtome section preparation. The Po signal was seen along the whole length of the spinal nerves, shown by the GAP-43 immunoreactivity. To compare the expression of Po and SI00, a marker for both CNS and PNS glia, Po expression was compared to the SI00 immunoreactivity on embryos of the same age. Po expression was tested on polyester wax embedded, microtome sectioned EI4 rat embryos and SlOO on cryosections of E l4 embryos which were PF fixed and sucrose protected, to obtain better antibody preservation. At El4, the DRG and the spinal nerves were positive for Po signal (Figure 4-5), while there was no SlOO immunoreactivity present (not shown). In the E l7 the distribution of Po signal in the DRG and roots was similar to that at E l4, and the Po level in the spinal nerves was higher, probably a combined effect of an increased number of Schwann cells and an increased level of Po in each cell. At this stage because of the further growth of the hindlimb it is not possible to include the entire length of sciatic nerves in a single section. GAP-43 immunoreactivity showed a large number of the putative cutaneous nerves under the dermis, which is reported by Reynolds and colleagues (1991). Some Po positive cells, although rare, were seen closely associated with some of these nerves (not shown).

4.2.5 Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization in the gut 4.2.5.1 Development of the gut The entire gastrointestinal tract with part of the parapharyngeal tissue attached was dissected. At E l2 the developing lung buds were located on two sides of the foregut and were used as a landmark. The nodose ganglia were located ventrally on two sides of the pharyngeal opening. The vagus nerves run from the nodose ganglia near the

140 dorsal surface and converge ventrally as they run into the esophagus/stomach mesenchyme. The stomach and the adjacent intestine were of similar sausage shapes and were distinguished by the presence of pancreas at their junction. The future intestine and colon were only two segments of tubes, with upper one of sausage shape and the lower one still partially fused to the umbilical cord. At E l3 the lungbud started to differentiate into several sacs. The stomach started to show the typical crescent shape, and grow much bigger in proportion to the growth of other parts of the digestive tract. Four twists of the digestive tract were seen in intestine portion of the gut, dividing the intestine into five portions. At E14 the lung buds developed into more complicated sacs, with each sac evolved into several smaller ones. The pharyngeal area has become muscular and bigger in proportion to other parts of the digestive tract. The wall of stomach has became much thicker due to substantial muscle development, and the typical asymmetrical shape was clearly observed. The intestines were held by a membrane while growth in length of each portion continued, thus producing the loop-shape of each portion, which were still distinguishable from each other. The 3rd portion grew much longer compared to other portions. The most distal portion of the duct, which was the shortest portion, grew much longer and remained straight as opposed to the other portions (Coventry et al., 1994).

4.2.5.2 Po expression in the gut: comparison to c-ret At E ll, a sub-population of neural crest cells which is c-ret positive have migrated into the circumpharyngeal area, seen in the whole mount El 1 rat (not shown). A sub­ population of p tubulin type 3 positive cells are also present in the forgut of El 1 embryo (Figure 4-6A). At the same stage little or no Po signal is detected in the circumpharyngeal area (Figure 4-3A and Figure 4-6A). At E12 a high level of Po expression was seen in the nodose ganglia, with the highest level in the roots and the trunks of the ganglia (Figure 4-6A). These stretches of Po positive cells which started from the nodose ganglia and ran uninterrupted in the esophagus were compact, as if the cells were trafficking through an invisible path. Judging from the location this pathway is the vagus nerves, which run from the nodose ganglia to the stomach to innervate the digestive system. Upon entering the stomach the expression was more scattered, but still in two streams in the upper half of the stomach. They became even

141 more diffuse in the lower half The expression could be seen in the second part of the future bowel, but not further than the cecum, the junction between the intestine and colon. Between the mid-otic placode and the caudal limit of the third somite, where the cardiac neural crest is located. Po positive cells were restricted to a small population of the crest. When examined in sections at higher magnification, the scattered cells in the mesenchyme around the primitive mucosa in the esophagus and stomach were polygonal mesenchymal-like cells and resembled the migrating neural crest in the trunk area (Figure 4-6B). Less intense levels of blue-purple staining were seen in individual cells scattered throughout the intestinal tract. Parallel c-ret ISH on preparations of the same age revealed that the Po positive crest derivatives had reached the same region of the gut as the c-ret positive neuroblasts at the same time.

At E l3 the Po was still expressed in the nodose ganglia as well as in the pathway along the esophagus. The highest expression of Po at this age moves down to the junction of the esophagus and stomach (Figure 4-6D). The signal was associated with the end of two vagus nerves as the nerves enter the stomach, and became more scattered in the body of the stomach. The purple dots of color production of Po signal are mostly in the first portion and the third portion of the intestine, and stopped at the cecum, the junction of the intestine and colon (Figure 4-6E). At this stage the c-ret positive neuroblasts have migrated over the cecum and reached the colon, the distal part of the digestive tract (Figure 4-6E insert). This is similar to the reported migration of DBH positive neuroblasts (Coventry et al., 1994).

At E l4 very little Po signal was detected in the digestive tract. Most of the expression was seen as scattered dots in the 3rd portion, which is the longest (Figure 4-6G). No signal was seen further than the cecum. A few odd positive spots could be seen around the pharyngeal area, probably within the vagus nerves. When E l3 and E14 guts were included in the same experiment thus enabling the direct comparison of the Po levels in single cells by the darkness of color precipitation, the signal in the intestine portion before the cecum at E14 was weaker than that at E l3, if not less in number. The very high level of expression seen in the top of the stomach in E l3 became very much weaker in E l4 (Figure 4-6F, compared to 4-6D). In parallel c-ret

142 experiments the number and level of c-ret positive cells were high at both ages. The down regulation of Po expression in the E14 gut occurs evenly in individual cells rather than declining in selected cells. In the adult no Po signal was detected in the plexus of the gut (Figure 4-61). After ISH, sections from different parts of the gut were post-stained with GFAP and SlOO antibodies to visualize the enteric and the peripheral glia. Of all the area tested, which includes esophagus, stomach, duodenum, intestine, colon, and the rectum, no Po signal was detected in any of the enteric glia, although it was clearly visible in peripheral nerves/ganglia outside the gut wall (Figure 4-6H, I).

In summary, a sub-population of migrating crest cells in the gut express Po as they migrate through the pathway of vagus nerves into the mid-gut, and the expression is down regulated by El4. The down-regulation persists through to adulthood. Po expression was not seen crossing the cecum to the hind gut. It is concluded that of all the circumpharyngeal crest in the hind brain/pharyngeal area the Po positive cells only consist of a small population of the vagal crest.

4.2.6 Po and type 3 ptubulin are expressed in the neural crest in vitro To be able to study the generation of Schwann cell precursors from the neural crest and to correlate expression of early markers such as Po and type 3 ptubulin, the caudal most part of the neural tube of E ll rat was enzyme dissociated, dissected, and plated on fibronectin coated coverslips, as described in chapter 2. Care was taken to dissect out the notochord and the neural tube formed by secondary neurulation. Neural crest grew out of the tube onto the fibronectin coated coverslip in 24 hours. These cells were recognized by monoclonal antibody 192 IgG, which recognizes p75NGFR (Figure 4-7A). The cells were located mostly in the dorsal aspect of the tube in the first 24 hours, but the cells grew out of both dorsal and ventral aspects of the tube in 2 days, as well as neurite outgrowth, as described in the literature (Bannerman and Pleasure, 1993). NT-3, NT-4 were added on top of the culture medium but no effect on the survival or death of the neural crest population was observed. Neural tubes were left in the culture all the time. In 2 days a group of cells expressed a low but distinct level of Po mRNA and protein, in the cytoplasm and in

143 the membrane, respectively (Figure 4-7CD). For the protein, some of the staining was seen around the nucleus, suggesting that the protein was being processed in the Golgi apparatus. There were different levels of staining within the population of Po positive cells (Figure 4-7D). These Po positive cells were of varied morphology, some were extremely flattened, tended to group together in pavements and were separate from the rest of the negative cells. Others have membrane ruffling on the edge of the flattened membrane, and the Golgi staining was more prominent. Cells which were Po positive were all in contact with the neurite outgrowth from the neural tube in some way or other. This was especially obvious at the contact line of patches with neurite contact or without. Those in contact with neurites express high level of Po while those without were significantly lower and could be regarded as negative. In the same culture system some cells already possessed TUJ-1 immunoreactivity at 24 hours and the cell shapes were invariably rounded (Figure 4-7B). A few TUJ-1 immunoreactive cells extended bipolar neurites. TUJ-1 was detected in mitotic cells, as described by Memberg and Hall (1994). (Figure 4-7B). No Po is detected in cells with rounded cell bodies.

144 4.3 Discussion In this chapter, the specific time and location of Po expression during mid to late gestation was studied, focusing mostly on derivatives of neural crest, with in situ hybridization and immunostaining methods on various ages of embryos and cultured neural crest cells. The results led to several major conclusions which will be discussed in more detail below; 1) Po is expressed in the migrating neural crest, but not in the newly emerging population. This adds direct evidence that migrating neural crest is a heterogeneous population. 2) Po is expressed in a sub-population of the migrating crest which does not include the melanocyte lineage. 3) Po positive cells in the ventral exit zone may not derive exclusively from the migrating neural crest. 4) the spatial distribution of Po strongly suggests it is expressed in immature peripheral glial cells. 5) Po is expressed in the glial lineage after the separation of neuron and glial fates. 6) Po is the known earliest gene to be expressed in the peripheral glial lineage. 7) Po is expressed in the migrating neural crest in the gut at the same time as the neurofilament positive vagus nerves innervate the gut, and one day after the first appearance of c-ret positive neuroblasts. 8) the transient expression of Po during embryonic development could be universal to PNS glia of different terminal phenotypes such as satellite cells and enteric glia, and early Po expression could mark the stage of a common precursor for different types of PNS glia.

4.3.1 Po is expressed in the migrating neural crest, but not in the newly emerging population. This adds direct evidence that migrating neural crest is a heterogeneous population A. Po is expressed in the migrating neural crest The Po signal was first detected in the ElO rat embryo in the migrating cranial neural crest population. At the trunk level, first of all, several approaches were used to confirm the anterior restriction of the Po expression along the embryo axis. This pattern was first observed in whole mount embryos of El 1 and 12 (as described in the Results section), followed by analysis of serial cross sections of several consecutive somites in the hindlimbs of E l2 embryos, where the pre-coalescing migrating neural crest could be best observed. When the extent of Po was plotted versus the body axis, a repeated pattern is noticed. On sections collected from the anterior part of the

145 somite, Po positive cells are present on both sides of the neural tube as well as extending laterally from the ventral exit zone (Figure 4-8A). This is followed by sections with less ventral extension (Figure 4-8B), then sections with Po signals only in the ventral exit zone and notochord (Figure 4-8C), when collected from more and more posterior positions. Each repetition is almost identical, with the lateral extension (in A) elongated further in more anterior somites. The length of one repetition (average 200 uM) is the same as the known rat somite length (Erickson et al, 1989). The fact that the Po signal extends more and more ventrally along the rostral-caudal axis is consistent with the observation that the neural crest migrates in a rostral-caudal gradient fashion (Erickson et al, 1989). The repeated pattern is the same as the typical segmented pattern of the migrating neural crest (Weston, 1963; Rickmann et al, 1985; Loring and Erickson, 1987; Erickson et al., 1989) and suggests that the Po is expressed in some cells within the migrating neural crest. Cells in the ventral exit zone were always Po positive except when the sections are exactly between somites. A series of consecutive sections through a whole mount embryo confirmed that the ventral Po positive dots correspond to the longitudinal line which runs parallel to the neural tube. The notochord was always Po positive. These cross sections represent a three dimensional distribution which consists of continuous longitudinal expression in the notochord and in the ventral root exit zone, and repeated patterns consisting of undulating distribution along each side of the neural tube. B. Po is not expressed in the newly emergent cells The overall pattern resembles the migrating neural crest with a small but significant difference: the newly emergent neural crest cells do not express the Po gene. This is apparent when the Po pattern is compared to the ErbB3 pattern. The ErbB3 was found to be expressed in developing Schwann cells and their precursors, and the migrating neural crest (Meyer and Birchmeier, 1995), but whether it is also expressed in neuronal or melanocyte precursors has not been described. In the anterior half of the somite ErbB3 was seen in the cells between the ectoderm and the neural tube and in the wedge between the neural tube, dermamyotome, and the overlying ectoderm, and this continuity stretches ventrally, first between the dermatome and neural tube, later near to the neural tube, and extends laterally at 2/3 length of the neural tube (Figure 4-4F). Po is evidently not expressed in cells located in the wedge between the neural

146 tube, dermamyotome, and the overlying ectoderm, where newly emergent neural crest cells are found. The fact that migrating neural crest cells only express Po after they migrate a distance from the dorsal midline demonstrates the distinct differences in the extent of fate restriction along the migrating pathway.

4.3.2 Po is expressed in a sub-population of the migrating crest which does not include the melanocyte lineage. The Po is not expressed in the melanocyte precursors of the migrating population. The precursors of melanocytes migrate in a non-segmented fashion (Weston, 1970; Serbedzija et al., 1989, 1990; Erickson et al., 1992; Richardson and Sieber-Blum, 1993). In the posterior half of the somite, BrbB3 is expressed in aggregated cells located at a wedge between the neural tube and dermamytome but above the sclerotome, which were reported to stain for HNK-1 as well (Bronner-Fraser, 1986). The fact that Po is not expressed in this group of cells demonstrates that Po is restricted to a sub-population of the neural crest which does not include the melanocyte precursors in the posterior half of the somite. In the anterior half, the Po positive population follows a different pathway than the melanocyte precursors. Po expression is very low in cells located between the dermatome and neural tube and above the sclerotome. This wedged space is the very spot where the melanocyte precursors were described to ‘sit’ before they go ahead to migrate along the basal lamina of the dermatome (Erickson, et al., 1992). Even in the more ventral position Po positive cells and the described melanocytes precursors have different adherence preferences. The Po positive cells were always very near the neural tube, while the melanocytes preferably migrate along the basal lamina of dermamyotome, disperse laterally and utilize the mesenchymal structures of the dissociated ventral part of the dermatome, and finally invade the ectoderm (Erickson, et al., 1992). The spatial segregation of the melanocyte migration pathway and the Po positive cells suggests that Po is not expressed in melanocyte precursors. Furthermore, experiments by Brennan et al. in our laboratory indicate that dissociated melanoblasts and melanocytes are Po negative, adding support to the point that Po is restricted to the sub-population of neural crest cells which does not include melanocytes. Po expression differs even within the migrating neural crest cells which take the ventral

147 pathway. Results from Dil-labeling experiments showed that migration along the ventral pathways appears to occur in two overlapping phases in the trunk (Serbedzija et al., 1990). Neural crest in the dorsal wedge moves first through the rostral sclerotome adjacent to the dermamyotome to populate more ventral structures such as the sympathetic ganglion, and then later, overlapping this population, a second phase of migration streams along the surface of the neural tube, opposite the rostral half of the sclerotome. As Po is only expressed in neural crest cells which are near to neural tube but not in those more lateral but still medial to the dermamyotome, it seems that the early migrating neural crest cells that have taken the ventral lateral pathway under the dermatome do not express Po until they reach their target site and colocalize with sympathetic neurons, while the late migrating neural crest which take the ventral medial pathway to populate DRG expresses the Po signal during migration, supposedly after the segregation of neuronal and glial lineages (see below).

4.3.3 Po positive cells in the ventral exit zone may not derive exclusively from the migrating neural crest One interesting difference between the picture of the Po signal and the whole migrating neural crest is that, while the neural crest migrates only via the anterior half of the somite, the posterior half of the somite was Po positive at two points which are the putative motor axon exit zones. In a cross section which cut through the hind limb of an E l2 embryo, this signal was seen as two dots on the sides of neural tube in a position two thirds ventrally. The presence of this Po signal in an area which neural crest avoids raise the question of whether these cells were derived from neural crest. It is known that a subset of neural crest migrates and contributes to cells that constitute the motor exit zone (Le Douarin et al., 1992; Golding et al., 1997). Judging from their location, Po positive cells that are associated with the motor axons as the axons exit the neural tube in the anterior half of the somite can only be from neural crest cells which migrate via the ventral medial pathway. But literature to date did not provide a definite explanation as to the origin of the Po positive in the exit zone in the posterior half of the somite. If the Le Douarin data (1992) also hold true in the posterior half of the somite, plus the well known fact that neural crest cells avoid the posterior half of the somite, the neural crest cells could only contribute to the

148 posterior exit zone by migrating rostro-caudally from the other half of the somite. However, HNK-1 studies of the neural crest showed that rostral-caudal migration only happens near the dorsal midline of the neural tube and when they reach dorsal aorta. Rostral-caudal mgiration at the ventral exit zone has never been reported (Bronner-Fraser, 1986; Loring and Erickson, 1987; Le Douarin and Dupin, 1993). Furthermore, it has been reported that some glial cells were derived from the neural tube (Lunn et al., 1987; Franz and Kothary, 1993). In grafting experiments in which the rhombomere 4 crest was replaced by midbrain and spinal cord neural crest, both neural crest populations contributed to the exit point in the heterotopic environment, suggested that the definition of the exit point lies primarily within the neural tube, not within the migrating neural crest cells (Niederlander and Lumsden, 1996). Therefore it is possible that specific groups of neuroepithelial cells differentiate at the presumptive exit-entry points and, apart from arresting the migrating neural crest migrating ventrally in the anterior half of the somite, also induce glial fate and Po expression in the exit zone in the posterior half of the somite.

4.3.4 The spatial distribution of Po strongly suggests it is expressed in immature peripheral glial cells In the trunk area the neural crest is known to give rise to cells of neuronal, glial, and melanocytic lineages (Le Douarin and Smith, 1988; Weston, 1991). With detailed analysis from whole mount and serial sections of the embryo, it could be concluded that the Po positive cells are indeed part of the migrating neural crest, and that Po positive cells are not found in the melanocyte lineage. The neural crest which takes the ventral-medial pathway, which we now know contains Po positive cells, gives rise to the DRG (Serbedzija et al., 1991). From this information it can be concluded that Po is specifically expressed in all or a sub-population of the neuron/glial lineage. To verify this hypothesis, the expression of Po was examined at another stage of differentiation , and also in the head area. In the front limb area of an E 12 rat embryo, where the DRG has already formed, Po is expressed only at the two poles of the olive-shaped DRG, and the extending tips of dorsal and ventral roots. This is similar to the pattern of ErbB3 expression in the front limb level of ElO mouse, which is equivalent to the E12 rat (Meyer and Birchmeier, 1995). Although it is not known

149 whether it is expressed by the immature sensory neurons in the coalescing DRG, ErbB3 is expressed in the migrating neural crest, Schwann cell precursors, and sub­ populations of immature Schwann cells (Meyer and Birchmeier, 1995). During development the glial cells in the sympathetic ganglion are described to have a similar polarized distribution to the newly formed DRG (Verdi et al, 1996). Later on, in E14 embryos Po expression in the DRG proper is very low or non-detectable, as opposed to the even distribution during the coalescing stage. The level of Po in the DRG remains the same in the adult (not shown). At E14 neurons could be distinguished from the satellite cells morphologically, and were never seen to be Po positive, and furthermore were never seen to express Po mRNA at any stage of development. The above all supports the idea that the expression of Po is spatially correlated to the glial lineage. In the E l2 head Po is not expressed in the area which would give rise to bone or cartilage, again restricting the expression of Po to the neuron/glial lineage. The expression pattern is restricted to the structures where the glial cells reside. This is shown by the restriction of Po in the head of an E12 rat embryo to all the recognizable cranial ganglia and nerves except the optic nerve, whose glial cells are of CNS origin. In the head of ElO and El 1 rat embryo when Po expression was first observed, Po was never seen in the epibranchial placode (see also chapter 5), which only gives rise to neurons but not glial cells (D'Amico-Martel and Noden, 1983; Le Douarin et al., 1986; Noden, 1993). The expression of Po was compared to the expression of Delta-1, which is a marker for placodally derived neurons, in the E l2 head area. The fact that these two markers were expressed in different anatomical structures in a different temporal pattern (ElO, 11), is consistent with the idea that Po is a glial marker, and a very early one. This observation of the absence of Po in several placodal structures which give rise solely to neurons reinforces the correlation of Po and the glial lineage.

4.3.5 Po is expressed in the glial lineage after the separation of neuron and glial fate The in vitro study of Henion and Weston (1997) shows that the separation of neuronal and glial fates takes place well before overt neuronal differentiation. In my in vitro study using the neural crest culture system, the initial Po signal was not detected until

150 at least 48 hours after plating. The Henion and Weston culture system is fairly similar to my culture, apart from the differences in species (rat versus quail). 30-36 hours after the initial outgrowth, the whole population of the neural crest is a mixture of the initial outgrowth and cells that migrate to the petri dish later. The sub-population of neural crest which migrates out 30-36 hours after the initial outgrowth consists almost only of neuronal and glial precursors and no bipotential progenitors. The degree of differentiation of the initial outgrowth which contains 50% bipotential progenitors at this time point is not clear, but it is very possible that the bipotential progenitor pool has run down to a similar proportion as in the later migrating population. Yet at this stage there is no Po detected in the whole population, suggesting that Po expression happens after the separation of neuronal and glial fates. Po signal was never detected in cells that have rounded up. Experiments by Dr. Calle in my laboratory using double staining methods shows that Po protein and type 3 p tubulin are expressed in different populations of the neural crest outgrowth. Po is not detected even in flat cells which are detected by antibody to type p tubulin. This strongly supports the notion that Po is expressed in the glial lineage after the separation of neuronal and glial lineages. The Po expression during the seperation of neuronal and glial lineages is summarised in figure 4-9.

4.3.6 Po is the known earliest gene to be expressed in the PNS glial lineage. Compared to the neuronal lineage, markers for the PNS glial lineage are fewer and less defined during the earliest stages of neuron/glial segregation and just after. Smith-Thomas and Fawcett (1989) reported the expression of Schwann cell markers SI00, NGF receptor and laminin, but no Po protein, in mammalian neural crest cell cultures, probably because of the inhibitory effect on Po expression of fetal calf serum and embryo extract and the sensitivity of the Po antibody used. 4B3 is a carbohydrate epitope which is reported to be expressed by the migrating neural crest (Cameron-Curry et al., 1991, 1993). Unfortunately no further study of 4B3 on neural crest has been reported to date. Reports by Dupin and colleagues (1990) were the first systematic research following the development and molecular phenotype of the glial derivatives with monoclonal antibody to the SMP antigen in a neural crest clonal system in quail. SMP is first present from an early developmental stage (E5 in quail.

151 E6 in chick), which is 2-3 days after the onset of neural crest migration and 5-6 days before the onset of myelination (Dulac et al., 1988, 1992). The expression of SMP is also found in mature myelinating and immature oligodendrocytes (Cameron-Curry et al., 1989) and the time course of SMP expression is later than Po (see below). The earliest Po protein expression detected by 1E8 monoclonal antibody, is reported in the migrating neural crest in chick (Bhattacharyya et al., 1991). Although not conserved in mammalian species, the Po epitope recognized by 1E8 was characterized as peptide rather than carbohydrate (Zhang et al., 1995). The Po mRNA expression in neural crest of rat is detected at a similar stage (Lee et al., 1997) as that reported for the chick Po protein (Bhattacharyya et al., 1991). However, minor differences of Po expression between these two species exist. Double labeling of 1E8 and HNK-1 in stage 19 chick showed that, apart from migrating near to the neural tube, Po positive cells are found beneath the dermamyotome, as well as in the mesenchymal tissue between the lateral end of the dermamytome and dorsal aorta (Bhattacharyya et al., 1991). The latter group of cells are migrating along the ‘ventral-lateral pathway’ to populate the sympathetic ganglia, as described by Serbedzija et al. (1989). In my study of rat the Po mRNA is restricted to the late-migrating cells which migrate via the ‘ventral-medial’ pathway to populate the DRG.

As for the molecular phenotype of the glial lineage in the rat model, Gal-C, SCIP (Oct-6/Tst-l) and PLP have been reported to be expressed before the onset of myelination at birth. Initial Gal-C synthesis is detected in pro-myelinating Schwann cells before the onset of myelination at E l8 and in the non-myelinating phenotype at postnatal day 19 (lessen et al., 1985). It was suggested that the Schwann cells start to express Gal-C at about the same time as cell division stops, in either the myelinating or at a later time in the non-myelinating cells. Another molecule expressed early is the transcription factor SCIP (Oct-6/Tst-l). Reports about the function and time course of expression are somewhat contradictory, but the earliest reported time of SCIP expression was by Blanchard and colleagues in Schwann cell precursors, in the sciatic nerves of E l2 mouse or E l4 rat (Jaegle et al., 1996; Weinstein, 1995; Bermingham et a l, 1996; Blanchard et al, 1996). It is difficult to conclude from the available data if Po expression precedes that of SCIP (Oct-6/Tst-l), since both are

152 expressed in the sciatic nerves of E14 rat (or equivalent stage of E l2.5 mouse), and the expression of SCIP (Oct-6/Tst-l) before this age has not been tested. The DM-20 transcript, an alternatively spliced form of PLP, is the dominant form during early embryonic development and is reported to be expressed in the CNS of E l0.5 mouse embryo (Timsit et al., 1992; Timsit et al., 1995). The expression of PLP/DM20 transcript has not been reported in detail in the PNS. However, the early expression pattern detected by enzyme reaction in a mouse carrying an defective PLP gene which is interrupted by the P-galactosidase suggests that PLP is expressed in the migrating neural crest and its derivatives (Ml Lee, unpublished data). This suggests that the Po is not the only myelin gene which is expressed in the neural crest population (see also chapter 5).

An important finding in my results is that Po expression is earlier than SI00 expression. Po is already expressed in the dorsal and ventral roots and spinal nerves in the hindlimb level of the E14 embryo, while my result show that the SI 00 is absent in the spinal nerves of the same area (Ml Lee, unpublished data). A similar time course of SI00 expression was reported in the mouse (Murphy et al., 1996). The Po protein is also detected by high sensitivity immunocytochemistry in rat E14 Schwann cell precursors, which lack SI00 in their cytoplasm (lessen et al., 1994; Dong et al, 1995; Gavrilovic et al., 1995). This suggests that Po expression precedes that of S I00, a conventional glial marker in the glial lineage. The significance of this finding is that the expression of Po is first detectable much nearer to the point when the glial lineage is generated from the neural crest.

Another interesting result is that Po gene is expressed in the glial lineage earlier than Krox-20 mRNA. As shown in the Results section, Po is already expressed along the length of cranial nerves and the migrating neural crest at the hindlimb level of E l2 rat embryo, while Krox-20 expression is only found in the entry/exit zone of cranial nerves but not more caudally. Po is expressed in Schwann cell precursors in the sciatic nerve but Krox-20 only appears after the transition of precursors to the Schwann cells (Topilko et al., 1994; Murphy et al., 1996). A semiquantitative RT- PCR study on developing mouse and rat nerves by Blanchard et al. (1996) showed a

153 similar time course for Po and Krox-20 expression. In this case the early Po expression in the neural crest and Schwann cell precursors cannot be regulated by Krox-20. Krox-20 is a zinc finger transcription factor which is required for the myelination of peripheral nerves (Topiko et al., 1994). In a mouse which carried a null allele in the Krox-20 gene myelination was arrested at a stage when the Schwann cells had established 1:1 relationship with the axons. The Po gene is down regulated by 20-35 fold in the mutant mouse (Topilko et al, 1994). On the face of it, the earlier expression of Po and the influence of Krox-20 on Po seems contradictory, but if we take into account that the level of early Po expression is much lower than the level when the Schwann cells are actively myelinating, we may conclude that the early Po expression is not myelination related, and not regulated by Krox-20.

4.3.7 Po is expressed in the migrating neural crest in the gut at the same time as the neurofllament positive vagus nerves innervate the gut, and one day after the first appearance of c-ret positive neuroblasts. The Po gene is first detected in the E l2 gut. The Po positive population moves distally along the future digestive tract at E l3 and the expression is quickly down- regulated during the next day, and remains inhibited in adulthood. This down- regulation of Po gene during development of gut has not been previously reported, although the absence of Po expression in mature enteric glia is reported in chick (Bhattacharyya et al., 1991). Comparison of the first appearance of Po and c-ret in the gut pathway shows that c-ret positive population has migrated into the esophagus while the circumpharyngeal area is almost Po negative, as shown in the whole mount E ll rat (Figure 4-3A). The vagus nerves, which are known to innervate gut via the same route as the enteric neural crest (Payette et al., 1984; Tucker et a l, 1986; Baetge and Gershon, 1989), are not projecting to their target yet at this stage. At E9.5 mouse, equivalent to E ll rat, the developing vagus nerves reach only to the level of the presumptive nodose ganglion (Baetge and Gershon, 1989). This demonstrates that the first population of neural crest migrates into the future digestive tract before the vagus nerves enter the same pathway. This first population are c-ret positive but do not have significant Po signal, and the Po signal is only detected one day later, when the vagus nerves enter the same pathway (Baetage and Gershon, 1989). The latter observation

154 could be interpreted as follows; the c-ret positive cell population migrates ahead of the Po positive cells, or this expression of Po is associated with the presence of vagus nerves or a combined effect of vagus nerves and c-ret positive neurons. As the Po gene is up-regulated by association with large diameter axons in the peripheral nerves, it is reasonable to suggest that Po expression in the migrating neural crest in the gut is also related to its association with the vagus nerves. Unfortunately no further experiment was done to support this hypothesis. Nonetheless, this system could serve as an ideal model for study separately the effect of close association with immature neurons or nerves in regulating early Po expression, since the c-ret positive, supposedly immature neurons and the vagus nerves could be separately depleted, by using c-ret knockout mice (Durbec et al., 1996) or deletion of post-otic placode- derived ganglia by administrating bisdiamine (Kuratani and Bockman, 1992).

4.3.8 The transient expression of Po during embryonic development could be universal to PNS glia of different terminal phenotypes such as satellite cells and enteric glia, and early Po expression could mark the stage of a common precursor for different types of PNS glia. Most of the main PNS glial types apart from teloglia and olfactory ensheathing cells, i.e., the Schwann cells, satellite cells, the enteric glial cells, have been directly shown to originate from the neural crest (Le Douarin, 1982; Dupin et al, 1990; Frank and Sanes, 1991; Le Douarin and Tiellet, 1973; Pomeranz and Gershon, 1990). Whether all these types of glia derive during development from a common precursor cell is still unsettled. Phenotypes of these cells are largely similar but distinct differences exist. Separate precursors for Schwann cells in the peripheral nerves and satellite cells were proposed, but recent research has suggested that differences in phenotypes were due to the influence of the microenviroment, and that extrinsic factors are constantly needed to maintain these phenotypes. Upon the loss of these factors the glial cells rapidly regress back to immature types, and dedifferentiate according to the new environment they encounter (Dulac and Le Douarin, 1991; Cameron-Curry et al., 1993; lessen and Mirsky, 1983; Woolf et al., 1992). This plasticity is characteristic of

Schwann cells. The mature phenotypes of Schwann cells regress to an immature phenotype following loss of axonal contact. When placed in contact with different

155 types of axon, these cells express molecules according to the assigned axon and not the one they previously contacted (Bray, et al., 1981; Bunge et al., 1990; lessen and Mirsky, 1992). In the chick PNS glial lineage, the enteric glia and satellite cells can express a Schwann cell phenotypic marker, SMP, when cultured in an environment similar to that of Schwann cells, even at a stage when the cells have undergone terminal differentiation (Dulac and Le Douarin, 1991; Cameron-Curry et al., 1993). The plasticity and interchange of phenotypes of these glial types suggests that these cells might originate from a common precursor.

Having failed to detect a common phenotype for immature precursors, Cameron- Curry et al. (1993) proposed a model in which glioblasts, which can be identified by an early marker, 4B3, give rise to SMP positive progenitors for the Schwann cell lineage and SMP negative enteric glial and satellite glia. My study shows that Po is a gene that is constitutively expressed in the PNS glial lineage (Lee et al., 1997) and is regulated in a very similar way to SMP in the chick. Furthermore, a new finding is that cells of different types of PNS glial express the Po gene transiently during development. A Po positive progenitor exists soon after generation from the neural crest, and this phenotype persists during migration and is only down-regulated after reaching the target. For example, the cells in the forming DRG, presumably the future satellite cells, express Po during the early stage of DRG formation, which is consistent with the report that satellite cells in DRG express Po during development but Po was nearly undetectable in adult life (Lamperth et al., 1989). The sub­ population of neural crest cells which contributes to the enteric nervous system expresses Po when it is migrating into the gut, but the signal is soon down regulated as the cells approach their final location. Some Po positive cells were seen under the dermis and were closely associated with the smaller branches of the cutaneous nerves at E l7, a time when innervation of the skin is most active (MJ Lee, unpublished data). Whether the absence of Po is due to an active inhibition of the microenvironment, as it was for SMP in satellite cells and enteric glia, or these Po positive cells died during development, can not be concluded from my data. But at least in the non-myelinating Schwann cell phenotype it is a case of active inhibition of the Po gene instead of failure to survive (Lee et al., 1997; also see chapter 3).

156 Therefore it is very possible that a Po positive, immature precursor gives rise to all PNS glial types, and they all retain the potential to regress to this immature, Po positive phenotype and re-differentiate to form any other PNS glial variants if put in appropriate environment (Figure 4-9). This model is not without precedent. In the sympathoadrenal lineage of PNS neurons, the sympathetic neuron. Small Intense Fluorescent cells (SIF) and chromaffin cells of the adrenal medulla, were the three phenotypes found. Their phenotypes are interchangeable when influenced by extrinsic factors, and they all transiently express CA (catecholamine) during early development, which can find its parallel in Po in the glial lineage.

157 Po mRNA expression during development M E11 E14 P4 300 bp

200 bp -^ 1 8 1 bp

100 bp

Figure 4-1 RT-PCR of Po in developing embryo at E ll, E14. Comparison with postnatal day 4 sciatic nerve. Note that bands of the correct size 181bp are detectable in the embryo at El 1 and El 4. Note that the loading of the cDNA is not equal (see text). Figure 4-2 At ElO, Po mRNA, revealed by DIG-iabelled in situ hybridisation, is seen in the developing otic pit (arrow) and also in migrating cephalic crest cells (arrowheads).

Figure 4-3 At El 1 (A) the otic vesicles (OV) are strongly Po positive and positive signal is seen clearly in the ophthalmic and submandibular branches of the developing trigeminal (V) nerves and within the ganglion, and also in the position of the developing acoustic/facial (VII/VIII) nerves. Wedge-shaped steams of positive neural crest cells can be seen in the trunk region down to the level of the 18-19^ somite. From the first to the 10-12^ somite position, strong Po expression representing glial cells associated with the emerging motor axons (arrows). (B) Section in the forelimb region of embryo double labelled for Pq and TUJ-1 to visualise axons. Pq positive cells are seen associated with the ventral root exit point.

Figure 4-4 At E12 (A) whole mount view of Po mRNA and (B) TUJ-1 labelling. At El 2 the otic vesicle (OV) and trigeminal ganglion (V) are strongly Pg positive, and the acoustic/facial ganglia (arrow) and nerves are also positive. TUJ-1 labelling is clearly seen within the trigeminal ganglion and nerves and the acoustic/facial ganglia and nerves. Pq positive cells can be seen in the developing glossopharyngeal (IX), vagus (X) and accessory (IX) cranial nerves. Pq labelling in the anterior somites now extends to the tail region and labelling in the notochord is clearly seen (arrowhead); (C) longitudinal section, showing the segmented nature of Pq expression down the length of the trunk. Pq labelling is confined to the anterior portion of the somites; (D,E) erbB3 mRNA expression at E l2 in the hind limb region. (D) Anterior portion of somite. Cells expressing erbB3 have a more widespread distribution than cells expressing Pg. In particular note erbB3 positive cells in cells between the ectoderm and neural tube (nt) in the most dorsal migrating crest pathways, and within the myotome (m); (E) in the posterior part of the somite note erbB3 in an aggregated group of cells between the neural tube and dermamyotome, but above the sclerotome, where Pq is never seen. (F-I) Pq labelling in sections through the front and hindlimb regions. (F) Section through the hindlimb region, anterior half of somite. Note that neural crest cells are more Pq positive ventrally, and that cells in the ventral root exit zone (arrow) are strongly P q positive. The neural tube (NT) is negative. Cells in the notochord (NC) are P q positive. Cells in the dorsal region, where the melanoblasts migrate, are Pq negative; (G) posterior region of somite. Pq labelling is confined to the ventral root exit zone and notochord; (H,I) sections through the hindlimb region, anterior half of somite. (H) Cells in the lower part of the condensing DRG are positive as are the developing ventral roots and spinal nerves (n); (I) higher power view showing condensing DRG with Pq positive cells around and within the developing ganglion. (J,K) Krox-20 mRNA labelling at E l2. (J) Hindlimb region. Note that Krox-20 is not seen in ventral root zone glial cells at a stage where these cells are strongly Pq positive (see F); (K) Krox-20 in glial cells associated with the cranial nerves in the rostral/head region. Note that, Pq mRNA is detectable in trunk glial cells before these cells become Krox-20 positive.

159 E1 0 E12

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In E14 various levels of Pq mRNA are expressed in regions of the PNS including the dorsal root ganglia (drg), dorsal roots (dr), ventral roots (vr), and sciatic nerves (n). The blue-purple signal in the dorsal and ventral roots is very dark, and signal was absent in the CNS. The signal in the DRG was of low level and sporadic. E11 B

TUJ-1 mBmmmm .

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Figure 4-6

P(3 expression in the enteric nervous system. (A) TUJ-1 positive neuroblasts are seen in the foregut region of an El 1 embryo double labelled with and TUJ-1. Note that no Pq positive cells are seen, (B) at E l2 Pq positive cells are seen in the region of the oesophagus. (C) In a whole mount preparation at E l2 streams of Pq positive cells can be seen emanating from the direction of the nodose ganglion (arrow) and migrating along the course of the vagus nerves. Pq positive cells are seen in the stomach (arrowhead). (D) At E l3 the highest level of Po expression is seen at the junction of the oesophagus and stomach. (E) Pq signal is also seen in the intestine (arrowed) but is not seen in the caecum (arrowhead) or beyond. At this stage c-ret labelling is evident beyond the caecum (insert). (F,G) At E l4 Pq expression is down-regulated. (F) Very little labelling is evident in the stomach. (G) In the hindgut, individual cells are much less strongly positive than at E l3, although their distribution is similar, i.e. no positive cells are seen in the caecum or beyond. This suggests that factors within the gut environment down-regulate P(j mRNA expression. (H,l) In the enteric plexuses of adult gut Pq positive cells are never seen. (H) GFAP labelling to show enteric glia within the enteric plexus of the gut. (I) No P(, labelling is seen with in situ hybridisation of the same section Figure 4-7 Expression o f neuronal and glial markers in neural crest cultures (A) Neural crest cell outgrowth at 24 hours, visualized by immunocytochemistry using monoclonal antibody to p75NGF-R. (B) Type 3 p tubulin expression in neural crest outgrowth 48 hours after plating. Cells which express this marker have rounded bodies and some are proliferating. In two days a sub-population of cells in the neural crest outgrowth expresses Po protein and mRNA. (C) Po mRNA visualized by NBT- X phosphate reaction. The mRNA is seen in the flattened cytoplasm. Most positive cells were in close contact with neurites which extend from the neural tube. (D) Po protein was visualized by a polyclonal antibody and second layer coupled to fluorescein. Some of the staining was seen around the nucleus, suggesting the protein is present in the Golgi apparatus. Different levels of staining within the population were observed.

163 Neural crest culture

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Figure 4-8 The periodic pattern of Po expression in the migrating neural crest. The extent of Po was plotted versus the longitudinal body axis. On sections collected from the anterior part of the somite, Po positive cells (shown as red dots) are present on both side of the neural tube as well as extending laterally from the ventral exit zone (A). This is followed by sections with less ventral extension (B), and sections with Po signals only in the ventral exit zone and notochord (C), when collected from more posterior position. NT; neural tube NC: notochord D: dermatome (adapted from Bronner-Fraser, 1993). Figure 4-9 A summary of Po expression during the generation of peripheral glial phenotypes. At the trunk level the neural crest cells give rise to melanocytes, neurons and glial cells. As the migrating neural crest cells are a mixture of bipotential progenitors as well as cells destined to a particular fate, for simplicity only the bipotential progenitor for neurons and glia is detailed. The bipotential neuron/glia precursors give rise to TUJ-1 positive neuronal progenitors as well as Po positive glial progenitors. Po is expressed in the glial lineage after the separation of neuronal and glial lineages, although whether or not PO positive progenitors retain the ability to regress to a bipotential progenitor remains a question mark. The Po positive glial progenitor gives rise to all PNS glial types. Po expression is complex. Basal levels seen in the glial progenitor, immature Schwann cells and immature enteric glia and satellite cells are down-regulated during differentiation. In the case of myelin forming Schwann cells, Po basal levels are strongly up-regulated by axonal signals. Different densities of hashed line indicates the Po levels.

166 Neural Crest StemCell

Other ! Bipotential progenitor N/G progenitor ceils

NRG

Neuronal Glial Progenitor progenitor

Immature Immature Immature hwann cell enteric Neuron Satellite cells glial cells tl

on-myelinatin Myelinating Satellite Enteric Schwann cells Schwann cell cells glial cells Chapter 5

The Expression of Pq in Placodes and Their Derivatives: The Inner Ear and Nose

168 5.0 Table of contents 169 5.1 Introduction 171 5.2 Results 175 5.2.1 Po expression in the trigeminal crest and trigeminal placode 175 5.2.2 Cranial neurons derived from the epibranchial placode do not express Po, as comparison with Delta-1 gene shows 176 5.2.3 Po is not expressed in the lens placode or its derivatives 176 5.2.4 The Po expression in the otic placode and inner ear 177 5.2.5 Comparison to other markers 179 5.2.6 The Po expression in the olfactory placode and its derivatives 180 5.3 Discussion 182 5.3.1 The Po is differentially expressed among neurogenic placodes. The differences are probably related to the ability to generate different cell lineages 182 5.3.2 The expression of Po and PLP in the otic placode suggests that neural crest and some placodes inherit a similar set of molecules that are differentially expressed during development 183 Table 5-1 Comparison of neural crest and neurogenic placode 183 5.3.3 Po is expressed early in the otic vesicle in the nonsensory epithelium, which includes everything except the sensory epithelium 184 5.3.4 Po expression precedes the invasion of melanocytes in the inner ear. Cells derived from the early Po positive cells in the non-sensory epithelium could be involved in ionic exchange 185 5.3.5 Po could function as an adhesion molecule by interaction with other adhesion molecules in the early development of otic vesicle 187 5.3.6. Are these Po positive cells of the same lineage as the peripheral glial cells? 189 Table 5-2 The earliest innervation of inner ear 190 5.3.7. Another Po mRNA species? 190 5.3.8 The Po expression during the development of olfactory system in rat embryo is similar in chick 191 5.3.9 The new discovery of Po expression in the adult olfactory nerves

169 suggests a different mechanism of regulation from gut and non-myelinated cells 192 5.3.10 A possible role for Po expression as permissive substrate for axons during early development and regeneration 193 5.4 Illustrated material Figure 5-1 Po expression in El 1 : comparison to Delta-1 194 Figure5-2 Po expression in otic vesicle: comparison to PLP/DM-20 194 Figure 5-3 Po expression in E l4 inner ear, serial sections 195 Figure 5-4 Po expression in utricle/cochlea/ganglion at E l4 196 Figure 5-5 Po compared to c-ret and BMP-4 in E l4 inner ear 197 Figure 5-6 Po compared to BMP-4 and serrate-1 in E l4 cochlea 199 Figure 5-7 Po compared to c-ret in E14 utricle and cochlea 199 Figure 5-8 Po expression in developing olfactory nerves 200

170 5.1 Introduction The placodes In chapter 4 the expression pattern of Po in the developing nervous system is described in detail and it is established that Po is an early marker of peripheral glia. This however does not explain the expression of Po in the otic placode seen from ElO onward in rat. The Po expression seen in the otic pit persists through the otic vesicle stage and continues to be expressed up to El4, the latest stage examined. A substantial part of this chapter is devoted to presenting this data. Another piece of data not discussed in detail so far in my study is the expression of Po in the olfactory system. Preliminary results gained from study of the embryos showed that, as the olfactory nerves project towards the olfactory bulb, their CNS target, a high level of Po expression was found along the length of the olfactory nerve. As introduced in Chapter 1, the otic vesicle, which the inner ear is entirely derived from, originates from the otic placode, and the olfactory epithelium and cells of olfactory nerves are believed to originate from the olfactory placode. Following the study of Po in neural crest derivatives, in this chapter it is felt that an extension to include the placodes and its derivatives is needed, as the otic vesicle and the olfactory epithelium are both assumed to be derived solely from neurogenic placodes. Both neural crest and placodes are capable of neurogenesis and contribute to primary sensory neurons and autonomic neurons in cranial ganglia. Furthermore, they share many similarities and probably evolved from the same structure during evolution (Mackie, 1995; Powell et al., 1996; Baker and Bronner-Fraser, 1997). Both structures originate from regions of columnar neurogenic epithelium lying between the neural plate and the surrounding non-neural epidermis. Both cell types undergo an epithelial-to-mesenchymal transition and delaminate from the neuroepithelium; both are capable of extensive migration. Their derivatives overlap to some extent in that both form sensory neurons, glia, neuroendocrine cells and cells secreting special extracellular matrices (for a review see Baker and Bronner-Fraser, 1997). The similarities are most obvious during the process of neurogenesis. As Po is expressed both in neural crest derivatives and placode derivatives, it is interesting to study the lineages of the Po positive cells in these two structures, in the hope that it will lead to a better understanding of the

ontogenic relationship of neural crest and placode.

171 In this extension of the study of Po to the placodes and their derivatives, it is intended firstly to examine whether the hypothesis that Po is an early glia marker could hold true when considering its expression in the placode. In other words, do placode derived neurons express Po? A second goal is to see whether the Po expression pattern suggests a hypothesis that a common lineage of neural crest and placode exists. Thirdly, does Po expression correlate with any molecular differences between different placodes? Lastly an attempt is made to further detail Po expression in individual placode derived organs, as well as to try to find whether this gives clues to their role in the organogenesis and function of the organ. A lot of emphasis is put on the inner ear, which expresses Po at a very high level during development in a pattern that is unique among other markers known to play important role in ear organogenesis.

The ear During the last few years the powerful tools of molecular biology have revealed a lot more of the mechanisms which control the development of the inner ear. A large number of molecules have been discovered to be expressed during the early stage of inner ear formation, a large proportion of which were first cloned in other organs, especially the nervous system (for review, see Torres and Giraldez, 1998). They can be roughly categorized into 4 groups depending on their possible function(s) in the inner ear. The first group are genes which are responsible for positional information, such as genes of the box family. The genes of the second group are involved in the process of cell differentiation during lineage separation, such as delta-1 and notch. The third group are lineage marker genes, which are only expressed after the specification of cell fate. Neuronal and glial genes are among members of this group. The fourth group are genes which function during the morphogenesis of the organ. There are two types of functions to carry out, one being generating the force to pull or repel neighboring cells to distort the shape of the organ, and the other one to programme cell death in certain areas to change the shape of the organ. Adhesion molecules are probably genes of the former function.

172 Recent discoveries in molecular biology of the inner ear have been concentrated on the first two types of genes, i.e., genes involved in location and genes involved in specification. Genes of the latter two types, i.e., genes responsible for morphogenesis and genes related to cell differentiation, have been less studied. Genes possibly belonging to these two types have been reported sporadically over the years, but no comprehensive view has emerged concerning the way in which they function during morphogenesis. In my study of the Po gene in the inner ear, apart from considering it as one of the placodes, I also wish to study its pattern during the development of the inner ear. By following the expression pattern of Po during inner ear development, I will try to assign Po to one of the four categories. The approach of correlating the pattern of gene expression and its function is not unprecedented. For example, in the inner ear, Fekete’s boundary theory (see 1.15.3.2 in chapter 1) states that the boundaries can be specified by limits of gene expression or a gradient of secreted inductive signal from neighboring tissue. The former predicts a visible boundary defined by gene expression. In the latter model, receptor expression on cells within the boundary is still needed for a cell to respond to the inductive signal. These two models both predict compartments defined by gene expression. Moreover, genes directly related to the specification and differentiation of cells are expressed before overt differentiation of structure and cell types, and usually have a more restricted pattern of expression, confined to a certain structure or type of cells. The pattern of genes expressed early in the otic vesicle could therefore, beside observations derived from mutations, give some hints about their functions.

The nose Another placode derivative that is Po positive are the developing olfactory nerves. The olfactory glia are a unique glial type. They are reported to possess phenotypes which resemble both that of Schwann cells and of astrocytes (Doucette, 1990). Recent reports show that Schwann cell-like and astrocyte-like cells of the olfactory ensheathing cell population can be distinguished by their low affinity NGF receptor and E-N-CAM immunoreactivity respectively (Barnett et al., 1993). A further study on a clonal line of olfactory ensheathing cells showed that the two sub-types could be

derived from the same lineage (Franceschini and Barnett, 1996). The Po expression in

173 the glial cells seems to be in line with the conclusion drawn from Chapter 4, that Po is an early glia marker. But several further questions need to be answered. Firstly, what is the exact cell population of Po expressing cells in the olfactory system. Is it confined to the Schwann cell-like cells, or is it in both sub-types? Do the Po positive cells originate from neural crest, or placode? The available results to date do not answer this question for certain, but a study starting from the embryonic expression certainly is a start toward the answer. Another observation is that Po expression is also found in the olfactory nerve during adulthood. This is interesting in that the olfactory nerves are unmyelinated, although ultrastructurally the arrangement of axons and Schwann-like cells in olfactory nerves more closely resembles that of Schwann cell precursors in the developing sciatic nerve (Barber, 1982). It is already known from the study in Chapter 3 (Lee et al., 1997) that Po is actively down- regulated in mature non-myelinating spinal nerves, so perhaps the lack of down- regulation of Po points to the fact that these cells represent a relatively immature cell population. The fact that olfactory nervous system is well known for its ability to regenerate, even in adulthood might be related to this relative immaturity (Barber, 1982; Graziadei and Monti-Graziadei, 1978 a, b, 1979). The significance of Po in this system certainly needs further exploration.

174 5.2 Results The general expression of the Po gene in rat during mid gestation stage was examined by in situ hybridization method using riboprobes labeled with digoxygenin. Whole embryos of ElO, E ll and E12 rat were used. The reaction color product for in situ hybridization was over-developed for further sectioning. For cryosections, whole mount embryos were gross-dissected to six or seven segments corresponding to different body axes, and tissues of known body axis were cross sectioned unless otherwise stated at 20\i using the sucrose protected, gelatin embedded cryosection method (see chapter 2).

5.2.1 Po expression in the trigeminal crest and trigeminal placode Po mRNA is first detected in the ElO rat. The area above the eye, where the future ophthalmic lobe/branch of the trigeminal ganglion/nerve is located, is Po positive (Figure 4-2). At El 1, the signal is still in cells above the eye. They aggregate nearer to the neural tube and cells of mesenchymal morphology spread along a pathway extending from the aggregation over the top of the eye. There is a brief but distinct gap of Po expression between the aggregation and the mesenchymal-like cell, which are TUJ-1 positive (Figure 4-3 A). These Po positive cells were not superficial and Po was not expressed in the ectoderm. In some experiments some cells located between the neural tube and the Po positive aggregation express the Po signal as well. To observe the expression of Po in the trigeminal placodes and their early derivatives, rat embryos were examined at ElO and E ll, concentrating on the head area. The fusion of the neural folds starts from the middle of the cranial-caudal axis and proceeds toward both ends. The earliest stage of this process could be seen in a murine embryo with 5-6 somite pairs, which is E8 in mouse and equivalent to an early ElO rat. The cephalo-caudal flexion of the embryo is still ventrally convex, ‘unturned’, at this stage. In an older ElO rat embryo with 9-11 somites the neural tube in the head area has already fused, and the embryo is dorsally convex, ‘turned’ (Brown, 1990). The neural tube closes during ElO in rat. As many studies of placode have been performed on amphibian and avian species (for example, Noden, 1978; D’Ami co-Martel and Noden, 1983; Le Douarin et al, 1986; Gallanger et a l, 1996; Stark et a l, 1997), and the records for the exact time point of placode formation in rodents are scarce, most

175 of the estimated placode formation time in the rat stated here is assumed, using the results from placode markers in avians. Comparing across species or strains by somite-stage is common and clearly superior to age or crown-rump length, although it is necessary to bear in mind that somites develop earlier and more rapidly in the chick than in rodents (Brown, 1990). At around the 8 somite pair stage in chick (equivalent to the 5-10 somite pair stage in rat) (Kaufman, 1990; Stark et al, 1997), expression of the placode marker Pax-3 was observed in the laterally extended ectoderm at the trigeminal placode level before it delaminates. Pax-3 positive placode cells start to invaginate and move away from the ectoderm after the neural tube closes in the head region at the 13-16 somite stage in chick (Stark et al, 1997). At the pre-delamination stage in rat (10 somite pairs, older ElO), Po is already present in the anlage of the ophthalmic lobe above the eye. This suggests that Po is expressed in the ophthalmic area before the neurons delaminate from the placode. A more detailed study is need to confirm the expression of Po in this area, but my results so far suggest that Po in the trigeminal area is restricted to the neural crest derived glia.

5.2.2 Cranial neurons derived from the epibranchial placode do not express Po, as comparison with Delta-1 gene shows. In the ElO rat embryo at the location where the epibranchial placode situated, no Po signal was observed. This suggests that the epibranchial placode derived neurons do not express Po when they delaminate from the placode. As Po starts to be expressed in the migrating neural crest only after migrating away some substantial distance from the initial delamination site, the Po expression at a later stage needs to be followed. Delta-1, a gene that is involved in lateral inhibition of neurogenesis in Drosophila, is expressed in the early placode derived cranial neurons in the chick (A. Graham, personal communication). To see if epibranchial placode derived neurons express Po after delamination, Po and Delta-1 gene expression in E ll rat were compared using whole mount in situ hybridization techniques. As indicated by Delta-1 expression, the epibranchial placode derived neurons were located near the opening of pharyngeal pouch and are on their way to their final destination (Figure 5-IB). In the same position there is no Po expression (Figure 5-IA), while at the same stage there is already a high level of Po expression among the migrating neural crest cells in the

176 PNS (see Figure 4-3A). This indicates that the early epibranchial placode derived cranial neurons do not express Po.

5.2.3 Po is not expressed in the lens placode or its derivatives There is no sign of optic development at the 0-6 somite pair stage during murine development (Brown, 1990). The optic pits are first observed in an murine embryo at the 8-9 somite pair stage as a depression in the still opening neural ectoderm. The optic eminence could be seen as protrusion from the side of the rising neural placode at the 9-11 somite stage. At somite stage 10-12 the initial cephalic site of neural fold fusion was seen. After the closure of the neural tube at the 20-25 somite pair stage the first branchial arch is formed and soon after this at the 25-27 somite pair stage the optic eminence could be distinctly seen above the first branchial arch. At the 30-35 somite pair stage the lens pit could be seen (Kaufman, 1990). At all these stages, which is equivalent to younger rat ElO embryo to E l2 embryo, no Po signal in the optic or lens placode or its derivatives was observed.

5.2.4 The Po expression in the otic placode and inner ear Po is expressed in the otic placode/pit at ElO rat, which has about 10 somite pairs. This Po positive structure is already in the process of invagination. The whole thickness of the convex part, together with a neighboring flat area around the invagination, is Po positive (Figure 4-2). The otic placode invaginates and forms a vesicle from Ell in the rat. Po is expressed nearly all over the vesicle at Ell (Figure4-3A and 5-1 A). As there is no distinctive morphogenesis before E l2, several markers that were reported to be specific to certain structures or cell types were compared to the pattern of Po. Digoxygenin-labeled riboprobes of BMP-4, c-ret. Delta-1, Serrate, Krox-20, Msx-1, FGF-3 were used. The body segment containing the otic vesicle and the first two branchial arches were dissected from E l2 rat embryo before fixation. Segments were processed in different containers with different probes in the same experiment. An ElO mouse embryo with a (3-galactocidase gene (p-gal) inserted under PLP/DM-20 promoter was immunolabeled in a whole mount preparation and anti-p-gal antibody was used to reveal the activation of PLP/DM-20.

After in situ hybridization or whole mount staining, the tissues were sectioned at 20-

177 50 fj.M horizontally using the sucrose protected, gelatin embedded cryosection method (see chapter 2). When roughly dividing the vesicle into quarters using the dorsal-ventral and medial-lateral axes, the Po signal, apart from being present in the endolymphatic duct, is located mostly in the dorsal-medial and ventral-lateral part of the vesicle. The expression of BMP-4, c-ret and PLP/DM-20 in the rat otic vesicle have not been reported previously. The BMP-4 expression in rat is similar to previously reported in chick (Wu and Oh, 1996). The Delta-1 and Serrate expression are similar to that previously reported (Myat et al., 1996; Lindsell et all, 1996). Unfortunately the embryos I used for Delta-1 and Serrate were older is development and thus not strictly comparable to the embryos used for Po expression (not shown). C-ret is expressed in the budding endolymphatic duct and in the medial-ventral and lateral-dorsal aspect of the vesicle, which is roughly reciprocal to the Po expression (not shown). The DAB reactions for the PLP/DM-20 signal were located in the dorsal-medial and ventral-lateral positions, similar to that of Po, but weremore restricted (Figure 5-2B). No Krox-20 and Msx-1 expression was detected in the otic vesicle at this stage (not shown). As no adjacent section to the Po one was hybridized with markers for the sensory epithelium, it is not feasible to suggest any correlation of any structure with Po expression at this stage. However, the tentative conclusion is that the overall pattern of Po expression is very different, if not completely reciprocal, to the expression of BMP-4, c-ret, delta-1 and Serrate, and is similar to the PLP signal. Although the specification of the cochlear duct to the ventral half of the vesicle and the semi-circular canals to the dorsal half of the vesicle already take place before this stage (Li et al., 1978), there is no telling whether the Po pattern is specific to any of the structures at this stage.

To further probe the expression of Po in the developing inner ear, the head of an E14 embryo was horizontally sectioned using the sucrose protected method (chapter 2). A series of adjacent sections were collected on adjacent slides for different probe processing. Every other section was hybridized with Po probe. Fig 5-3B is a representative section that slices through the endolymphatic duct, the semi-circular canals and part of the utricle. A series of neighboring sections confirms the identities of these structures (Figure 5-3). Po is expressed in the base of the endolymphatic

178 duct, and in parts of the semicircular canals. Fig 5-4 is a representative section more anterior to Fig 5-3. The cochlea, part of the utricle and the semicircular canals are shown. In addition, the acoustic ganglion, which sits very near the cochlea, is Po positive. Like DRG of the same age the motor roots of acoustic ganglion which project toward hind brain and the sensory roots that innervate the inner ear are highly Po positive. Sporadic Po positive cells could be seen within the ganglion. One section of the ganglion that sits nearest to the cochlea, however, is completely devoid of Po positive cells except at the periphery (Figure 5-4). In the cochlea the pattern of Po expression is restricted to only half of the section and there is a very sharp boundary of expression and non-expression (for example. Figure 5-4, 5-6). At this stage the acoustic nerves have started to innervate the cochlea (Von Bartheld et al., 1991). The sensory epithelium, which is near to the ganglion and is supposed to be innervated, is serrate and c-ret positive but Po negative (Figure 5-6, 5-7). Within the Po positive patch there are different levels of expression, with the highest levels usually seen in cells facing the lumen, but in some areas the signal is found all through the thickness of the cross section (for example, see Figure 5-6C).

5.2.5 Comparison to other markers The Po pattern is unique when compared to other markers. In a series of parasaggital sections of the cochlea, Po expression is compared to adjacent sections hybridized with BMP-4, a putative marker for the sensory organs, and with c-ret, the marker for supposedly neuronal cells (Figure 5-5). The patterns of BMP-4 and c-ret are similar, which agrees with the idea that they mark cells of the sensory area. The Po expression is almost complementary to the other two markers, suggesting that Po is expressed in the non-sensory component of the inner ear (Figure 5-5). In the cochlear area, adjacent cross sections were hybridized with Po and other sensory markers as a comparison. Again the BMP-4 is complementary to Po expression. Serrate-1, a ligand for notch and suggested to play an essential role in neurogenesis of the sensory cells in the inner ear, was also compared to Po. The expression of Serrate-1 is restricted to the sensory epithelium of the inner ear (Whitfield et al., 1997). Again the patterns are complementary to Po expression in the cochlea (Figure5-6). 192 IgG, which is a

monoclonal antibody p75NGF-R, was tested in this experiment to examine the

179 innervation of the inner ear from the acoustic ganglion. While the ganglion and nerves were highly NGF-R immunoreactive, there is no signal within the epithelium of the inner ear (not shown). This result is different from the report by Von Bartheld et al. (1991). The absence of NGF-R signal in the epithelium could be due do either a difference in the level of mRNA (Von Bartheld) and protein level (MJ Lee, unpublished), or difference in sensitivity of the methods in detecting the lower level in the epithelium. In the utricle/saccule region, Po pattern is compared to c-ret. Two adjacent parasaggital sections of this region were hybridized with Po and c-ret. While c-ret only marks a small area of the utricle, which is supposedly the sensory area, Po covers a large area. Again the pattern is reciprocal, and the boundary between expression and non-expression is sharp (Figure 5-7).

There are no Po positive cells outside the structure of inner ear apart from those associated with the acoustic ganglion and nerves. In the cross section of cochlea, Po was only observed within the cochlea and not in any surrounding mesenchymal tissue, where the neural crest derived migrating melanocytes are located (Kitamura et al., 1992; Steel et al., 1992; Nakayama et al., 1998), which suggests that the Po positive cells are not from the migrating cranial neural crest. Unfortunately there is no reputable marker specific for structures within the nonsensory epithelium such as the stria vascularis or Reissner’s membrane at this early stage, and markers for the adult structure were not tested. Overall, Po expression is restricted to the non-sensory epithelium, the boundary between expression and non-expression is sometimes very sharp, and it covers all the area apart from the sensory area. Po expression could be confined to the stria vascularis and Reissner’s membrane or be more widespread and include more than these two structures.

5.2.6 Po expression in the olfactory placode and its derivatives The olfactory placode is formed at the 25-27 somite pair stage in mice. The olfactory pit is formed at the 30-35 somite pair stage in E l2 rat (Kaufman, 1990), which in turn gives rise to the olfactory epithelium. The olfactory placode and epithelium were studied in rat E l2 to E l4. The heads of E l2 and 13 embryos were dissected, and a coronal section made between the telencephalon and metencephelon. Some of the

180 tissues were sliced in half at the midline of the body. These tissues were processed whole mount for Po. For E l4 embryos, cross sections of the head were taken using the sucrose protected method (chapter 2), hybridized with Po and immunostained with 192 IgG antibody respectively. For adult, a pregnant rat that was sacrificed for the embryos was decapitated, the skull opened and the posterior part of the brain and cerebellum was removed before dissecting the olfactory nerves. Bundles of olfactory nerves medial to the lamina cribrosa and projecting toward the bulb were dissected. The olfactory nerves were buried in a muscle layer and were not ensheathed by connective tissue. These nerves and tissue from the olfactory bulb encapsulating layer were dissected and teased on TESPA coated slides as described in chapter 2. Po is not expressed in the olfactory placode at El 1, neither is it expressed in the E l2 olfactory pit, when examined whole mount or as dissected tissue. Po is first detected in the olfactory area at E l3. The signal is of higher level in the back of the olfactory epithelium. The signal persists along the projection. The signal disperses to cover a crescent shape around the olfactory bulb before it enters the CNS, as the olfactory nerves do. No experiment was done to examine the Po signal in the area where the nerves enter the lobe (Figure 5-8A). In sections of E14 olfactory epithelium, NGF-R immunoreactivity was seen in the periphery of the epithelium (not shown). The olfactory nerve bundles were NGF-R reactive as well. At E14 the Po was seen as dots at the periphery of the olfactory epithelium but not within it (Figure 5-8B). The teased adult olfactory nerves and cervical sympathetic trunk were hybridized with Po in the same experiment for a direct comparison. The adult olfactory nerves were positive under conditions whereas there is no Po signal in the adult cervical sympathetic trunk, which is 95% non-myelinated except in the occasional myelinating fibre (Figure 5-8).

181 5.3 Discussion The study in this chapter is an initial step toward understanding of the behaviour of the Po gene and reveals a possibly undiscovered function of this gene which has been shown to be very important in other cell types. A similar theme in Chapter 4 and 5 is to explain a gene’s behaviour and function, and possibly the way genes organize each other to achieve morphogenesis, by the expression pattern of this gene and its relation to the pattern of other genes. My study of Po in the placode derivatives, especially in the otic and olfactory placode, concludes as follows:

5.3.1 Po is differentially expressed among neurogenic placodes. The differences are probably related to the ability to generate different cell lineages. There are several differences between neurogenic placodes. For example, the olfactory and otic placodes give rise to more than one cell type. The otic placode gives rise to neurons, the sensory receptors (hair cells), non-sensory cells in the sensory organ, the epithelium lining the fluid labyrinth, and probably the bone structure surrounding the fluid labyrinth. The olfactory placode gives rise to the olfactory neurons and the olfactory ensheathing cells, while epibranchial and trigeminal placodes give rise solely to neurons. Secondly, the otic placode might utilize a different cell-cell or cell-matrix interaction from other placodes to provide the mechanical force to form a pit (Legan and Richardson, 1997). The expression pattern of N-cadherin in the nasal placode and optic vesicle is different from that of the otic placode (Duband et al., 1988; Hatta et al., 1987), and the actin cytoskeleton is not directly involved in otic pit formation (Hilfer et al., 1989). A recent report by Torres and Giraldez (1998) proposes a model in which specific combinations of genes rather than single gene expression appear to be characteristic for each placode. They suggest that transcription factors of six. Pax, dix gene families might contribute to the combinatorial codes. Although evidence is still incomplete, a trend toward different molecular compositions in different placodes is observed. That Po is expressed in the otic and olfactory derivatives and not expressed in the epibranchial and trigeminal placodes is another difference between the neurogenic placodes and provides another evidence that the molecular composition of placodes is different.

182 5.3.2 The expression of Po and PLP in the otic placode suggests that neural crest and some placodes inherit a similar set of molecules that are differentially expressed during development. Recent reports agree with the hypothesis that the neural crest and at least some placodes might be lineage or evolutionarily related. The existence of peripheral neuroendocrine cells associated with the dorsal strand, and probably in the floor of the pharynx, of the acidian, a representative species of the urochordata, showed that these migratory neuroendocrine cells might be the evolutionary ancestors of both placodes and neural crest cells (Mackie, 1995; Powell et al., 1996). Furthermore, the neural crest and placodes share similarities during development and their derivatives overlap to some extent, as described in the Introduction in this chapter. In a way the neural crest and otic placode behave fairly similarly, as examplified by the process of neurogenesis. As both structures differentiate, parallel components can be found in both (see table 5-1)

Table 5-1 Comparison of neural crest and neurogenic placode

parameter neural crest (e.g., DRG) otic placode Site of emergence closure of neural tube thickening of body wall Induction factors ectoderm and the tube ectoderm and the neural tube Mechanism of emergence delamination same Migration pathway top of tube to DRG placode to ganglion CNS target dorsal horn acoustic and vestibular nuclei Lineage specification during migration in placode/migration Formation of the nerve projection toward CNS projection toward CNS Aggregation of neurons DRG acoustic-vestibular ganglion The glial cells neural crest derived same

Using the PLP/DM-20 promoter-p-gal system, the PLP/DM-20 gene is detected in the migrating neural crest and the derivatives, the sub-ventricular epithelium, as well as in the otic vesicle as described above (M. J. Lee, unpublished). Tyrosine kinase

183 receptor ErbB3, which is also strongly expressed by neural crest cells and by Schwann cell precursors and Schwann cells, is expressed in the otic vesicle and inner ear as well (Meyer et al, 1997). The appearance of Po, PLP/DM-20 and ErbB3 genes in migrating neural crest and in some placode structure(s) suggests that these genes are probably specific to a family of cell lineages, i.e., the placode/neural crest/neural epithelium rather to only to the neural crest derivatives. This is consistent with a hypothesis that Po positive cells in the ventral and dorsal exit points originate from neuroepithelium rather than from neural crest (see Chapter 4, Discussion section). It also accommodates the facts that Po is expressed in immature Schwann cells, in the olfactory ensheathing cells, believed to be a new type of glial cells, and in the cells of the non-sensory epithelium of the inner ear, which is placode derived.

Evolutionarily, Po resembles an ancestral immunoglobulin gene superfamily member, and by gene duplication, this molecule might have yielded very different types of contemporary immunoglobulin-like molecules in the nervous system (Lemke et al, 1988; Williams and Barclay, 1988). On the other hand, traditionally it has never been uncovered anywhere except in myelinating cells, a uniquely vertebrate specialization (Trapp et al, 1988; Mirsky et al., 1980), suggesting that Po is evolutionarily as new an invention as the myelin sheath itself. The expression of Po gene outside myelinating cells in the notochord and in the otic placode therefore alleviates the paradox of a molecule possessing ancient molecular features while the expression pattern suggests a relative recent appearance. Another implication from Po expression outside the PNS glia lineage is that the expression is not a prerequisite for a glial fate. The expression of Po gene is therefore a consequence of the choice of glial fate. Anyhow, this is not contradictory to the conclusion in chapter 4 that Po is a marker for the early glia cells. The expression of the non-glia Po can be morphologically and temporally distinct from the glial Po, and therefore does not interfere with the representatives of Po marking the entry to the PNS glial fate.

5.3.3 Po is expressed early in the otic vesicle in the nonsensory epithelium, which includes everything except the sensory epithelium.

184 My study of Po expression in the early development of the inner ear shows that Po is expressed early in the otic vesicle in the nonsensory epithelium, which comprises everything except the sensory epithelium. At E l2 Po is expressed in dorsal-medial and ventral-lateral aspect of the otic vesicle, which include otic vesicle parts that later develop into semicircular canals, cochlear duct and utriculosaccular space (Li et al, 1978). At E14 the expression pattern of Po is complementary to the patterns of sensory epithelium markers, which include BMP-4, Delta-1, Serrate, and c-ret. As introduced in chapter 1, BMP-4 is established as a marker for the sensory epithelium in the chick (Wu and Oh, 1996). Results presented here show that the pattern of BMP-4 in rat is similar to that in the chick. Delta-1 and its homologue Serrate (Jogged-1) are suggested to be involved in the neurogenic process as well as in the specification of sensory hair cells versus the non-sensory supporting cells of the sensory epithelium (Whitefield et al, 1997). My results using Delta-1 and Serrate (Jagged-1) probes agree with what was reported in the chick (Myat et al, 1996; Lindsell et all, 1996). The patterns of these markers for the sensory epithelium are reciprocal to the pattern of Po. The c-ret expression was reported in the chick as confined to the dorso-lateral quadrant, and was interpreted as an area where the semi­ circular canals arose (Robertson and Mason, 1995). My results show that c-ret expression of inner ear during morphogenesis is broadly similar to those of BMP-4, Delta-1 and Serrate. Being a member of the receptor tyrosine kinase family and being important in the neurogenesis of the enteric nervous system, c-ret probably plays a role in the neurogenesis or sensory patch formation, or at least is an appropriate marker for the sensory area. Again the Po expression is complementary to c-ret, arguing that the Po is in the non-sensory epithelium.

5.3.4 Po expression precedes the invasion of melanocytes in the inner ear. Cells derived from the early Po positive cells in the non-sensory epithelium could be involved in ionic exchange. Immunochemical and ISH experiments using recently identified melanoblast markers such as MEBL-1, TRP-2/DT and mift have shown that the melanogenic subpopulation of cranial neural crest cells migrates directly into their final destination

in the inner ear (Kitamura et al., 1992; Steel et al., 1992; Nakayama et al., 1998).

185 Before they invade the inner ear, the mift positive melanoblasts are seen among the migrating cranial neural crest. They were located in the surrounding area outside the cochlea at E l4 and are seen in the epithelium of the inner ear of E l6 rat (Nakayama et al., 1998). In the adult the melanocytes are located in the non-sensory epithelium (Conlee et al., 1989). Po, on the other hand, is expressed from ElO onward in the rat. It is not expressed in the mesenchymal tissue outside the cochlea at E l4. Therefore the Po expressing cells and the melanocytes of inner ear are probably two different populations, and the Po expression in the otic vesicle precedes that of melanocyte markers.

The non-sensory epithelium of the inner ear is known for its function in maintaining the high and positive ion composition of the endolymphatic fluid. The melanocytes located in stria vascularis are believed to play an important role in ionic exchange, based on the fact that mutations resulting in lack of melanocytes in the inner ear lead to abnormal endolymphatic potential and therefore deafness (Steel and Brown, 1994). However, recent studies demonstrate that the melanocytes are only a small proportion of the cellular structure responsible for the ionic exchange of endolymphatic fluid, as recent studies on gerbils demonstrate (see below). The nonsensory epithelium consists of many different cell types and capillaries embedded in the modiolus bone (Spicer and Schulte, 1998; Sakaguchi et al., 1998). Following the opening of channels in hair cell stereocilia to generate electric stimuli, excess K is taken up by Deiter cells and passed through an array of ion exchange cells in the non sensory epithelium, and recycled back to the endolymph (Schulte and Steel, 1994; Spicer and Schulte, 1994, a, b, 1996; Kikuchi et al., 1995). is also actively transported from the perilymph in the scala tympani and scala vestibuli by a chain of different types of fibrocytes in the lateral wall and spiral ligament back to the scala media (Spicer and Schultte, 1988; see Figure 1-4). As is known, the mature Schwann cells, in which Po is constitutively expressed, function to maintain the ionic balance of the axon, an interface between two very different ionic potentials (Barres, 1990). They also express voltage- dependent sodium and potassium channels (Chiu et al., 1984). The mechanism of ion balancing used by cells in the non-sensory epithelium and by the Schwann cells are therefore similar in a way. Hence it makes sense to propose that some cells in the

186 non-sensory epithelium responsible for ionic balance are possibly the derivatives of Po positive cells seen in the embryonic stage.

Expression of ion transporters shows a differential pattern between the cells of neural lineage and cells of the non-sensory epithelium of the inner ear. Immunohistochemistry and ISH experiments using antibodies and probes specific to different isoforms of Na, K-ATPase showed that aiP 2 isoforms were present in the stria vascularis, Reissner’s membrane and dark cells of the vestibule, while cells of the neural lineage of the inner ear, including the hair cells, support cells, the spiral ganglion and its nerves, express isoforms (Ten Cate et al., 1994). Na-K-Cl

CO transporter expression shows a high specificity to the ion balancing epithelium as well (Sakaguchi et al., 1998). A detailed study of the co-expression of these two ion transport mediators and Po expression would firstly establish whether the early Po expression in the inner ear is restricted to the non-sensory epithelium, and secondly, whether there is high cellular correlation between the Po positive cells and ion- transporter. It would also be interesting to find out which type (non-sensory cell or neural cell) of Na, K-ATPase these Po positive cells express. In vitro culture (if possible) of these Po positive cells and a comparison of the molecular phenotypes to the Schwann cells in the PNS wold also give information about the identity and probable function of these cells.

5.3.5 Po could function as an adhesion molecule by interaction with other adhesion molecules in the early development of otic vesicle Po is expressed at a time when the inner ear is undergoing a large amount of morphogenesis. If indeed Po is expressed at the protein level and plays some role during morphogenesis, it is possibly very different from other genes that define the location or axis of the inner ear such as the homeobox genes, or genes involved in the generation of sensory organs such as BMP-4. A very distinct feature of the Po molecule is its adhesion ability. As is known, basic Po function during the much more studied myelination is based on its adhesion ability. It is therefore feasible to suggest a role for Po based on its adhesion ability during inner ear development. Molecules

involved in cell-cell interaction reported so far to be expressed in the otic vesicle

187 include NCAM, and NgCAM, LI, HNK-1(CD57) and CD 15 (Richardson et al, 1987; Meyer Zum Gottesberge and Mai, 1997; Legan and Richardson, 1997). The expression patterns of these molecules are up- and down regulated at different times and locations within the ear, and the pattern for Po is different from all these molecules. The biological significance of these patterns remains elusive. Expression of LI was examined from mouse E ll onward, and a role in the innervation of the vesicle was suggested (Mbiene et al., 1989). The down-regulation of CD 15 was correlated to the maintenance of the maturity of the structure, and the expression of HNK-1 was related to the ingrowth of nerve fibres (Meyer Zum Gottesberge and Mai, 1997). Richardson and colleagues (1987) proposed that the collective pattern of CAMs in the inner ear was correlated to border formation. However, a recent report by Wu et al. (1998) suggests that the anteior-posterior axis of the sensory position is specified earlier than E2.5 (otic vesicle stage), and the dorsal ventral axis of sensory organs and nonsensory organs is probably specified soon after (Van der Walter, 1983). In other words, the specification of the sensory area is probably complete after the vesicle stage, and the adhesion molecules expressed at and after this stage should be playing other roles rather than directing location information. The vesicle probably utilizes the adhesion molecules to achieve the complex shape of the inner ear by providing the physical force for holding together or pulling apart, via homophilic interaction of Po molecules or heterophilic interaction with other adhesion molecules, or even with the extracellular matrix, much like what happens during myelination. It might even be aided by programmed cell death to shape the inner ear. Cell death was reported recently in the neck-like structure or fusion plate of the inner ear in the chick (Fekete et al., 1997). Interestingly, in my study most of these areas lack Po expression. A detailed study of the adhesion molecules, especially Po, and the correlation with apoptosis, would probably give a better picture as to mechanism of the morphogenesis in the ear.

One question is not easy to answer when considering a role for Po in morphogenesis as an adhesion molecule: why does the gross anatomy of the inner ear look normal in the Po knockout mice (MJ Lee and E. Calle in collaboration with Prof. R. Martini, preliminary observations)? It is possible that the role of Po is minor or redundant, as

188 many other adhesion molecules are present in the inner ear. Another possibility is that the abnormality is subtle and was not described due to a bigger interest in examining the defect in myelination and in the ganglion of the acoustic nerves (Giese et al., 1992).

5.3.6. Are these Po positive cells of the same lineage as the peripheral glial cells? Some molecules which are expressed in mature Schwann cells are shown to be expressed in the mature inner ear. These include S I00 (Foster et al., 1993, 1994; Igarashi et al., 1991), HNK-1 epitopes (Meyer Zum Gottesberge and Mai, 1997) NGF-R (Von Bartheld et al., 1991), ErbB-3 (Meyer and Birchmeier, 1995), NCAM, NgCAM, LI (Richardson et al., 1987) and PLP (M-J Lee, unpublished). This thus raises a question: is the Po observed in the otic vesicle present in immature glial cells? The earliest cells to delaminate from the otic vesicle are the future sensory neurons of the Vlllth ganglion (Torres and Giraldez, 1998; Legan and Richardson, 1998). It is possible that the cells that delaminate from the otic vesicle contain committed glial precursors, although a study in the chick-quail system concludes that no glia cells are of placode origin apart from olfactory ensheathing cells (Le Douarin et al., 1986; Baker and Bronner-Fraser, 1998). However, Po is expressed within the otic vesicle and developing inner ear subsequent to this delamination. This argues against a purely glial fate for Po positive cells within the ear after the delamination. Later on the innervating nerves are a possible source for cells of peripheral glial phenotypes. The Schwann cell-like cells could migrate along with the acoustic nerves and invade the inner ear. However, the known molecular phenotypes of the developing inner ear argue against the possibility that the initial Po positive cells come from the innervating nerves. The earliest innervation of inner ear, demonstrated by immunoreactivity of LI, HNK-1, NGF-R, PSANCAM (see table 5-2), is between E12 and 16 in rat, E12.5-13 in mouse, and E4 in chick. Dye O injection experiments confirmed that the immunoreactivity of markers correspond well with the time of nerve fibers penetrating the presumptive anlage of the sensory organ at E4 chick (Von Bartheld et al., 1991). So the time the nerves penetrate the sensory epithelium is later than the initial expression of Po in the otic placode/vesicle, which is ElO onward in rat. Therefore, the early expression is not associated with acoustic nerves.

189 Table 5-2 The earliest innervation of inner ear

otic vesicle innervating nerve reference LI (mouse) ElO negative, starting at E l3 Mbiene et al., 1989 starting at El 1.5 HNK-1 (mouse) absent at El 1.5 starting at E12.5 Myer zum Gottesberge and Mai, 1997 NGF-R (chick) absent at E2.5 starting at E4-4.5 van Bartheld, 1991 patchy in E4 NGF-R (rat) positive at El 1,E 12 starting at E l6 van Bartheld, 1991 not in nerve

5.3.7. Another Po mRNA species? There is a possibility that the early Po signal detected in rat represents a similar but distinctly different mRNA specie(s). The riboprobe used in the in situ hybridization method for embryos is routinely hydrolyzed, and the lack of apparent signal in whole mount embryo when unhydrolyzed probe is used is interpreted as the inability of the longer probe to penetrate the tissue. The lack of apparent signal with whole length probe does not include sections of E l4 embryos, where the Po mRNA is present in Schwann cell precursors in the embryonic spinal nerves. Therefore it is possible that some mRNA similar to the Po gene could not be detected with unhydrolyzed whole length Po and could only be detected with hydrolyzed Po in early embryos of E ll or 12. More than one Po mRNA have been reported in vertebrate species. In trout two alternatively spliced forms of Po, IP-1 and IP-2, are expressed in the CNS and other similar two forms, IP-C and IP-D, have been detected in the PNS (Stratmann and Jeserich, 1995; Barbu, 1990). Only one single mRNA in chick and rat was found in poly-A RNA, and the several reported in chick total RNA were probably an artifact from 28S rRNA (Zhang et al., 1995; Barbu, 1990). A blotting experiment using only the otic placode tissue before the innervation of acoustic neuron, thus prevent neural crest derived glia contamination, would give an answer. The early expression in the otic vesicle and in notochord apparently is not regulated by axonal signals. After denervation or when cultured without neuronal signals, Schwann cells are capable of expressing basal levels of Po mRNA, suggesting that this level of mRNA expression in Schwann cells is independent of axonal regulation (Poduslo et al., 1985), while Po protein in this situation is destined for lysosomal degradation as soon as it is produced (Brunden, 1990). If the early expression in E ll and 12, especially in the

190 otic vesicle, is indeed of a different mRNA species, it is possible that the functions and the mechanism of the Po-like molecules are very similar to that of Po molecules, based on the observation that the non-sensory epithelium of the inner ear expresses high Po (or Po-like) mRNA and probably carries out functions such as modulating shape during organogenesis and ionic balance (see 5.3.4 and 5.3.5).

5.3.8 The Po expression during the development of olfactory system in rat embryo is similar in chick. Using Po riboprobe to detect the Po signal, I found that the olfactory nerves expressed Po from E l3 onward. This expression carries through and is not down-regulated in adulthood. The study by Gong and Shipley (1996) on the developing rat olfactory nerve shows that GAP-43 positive olfactory nerve has already project toward their CNS target at E l2, presumably from the primary olfactory neurons situated in the epithelium. Only one day later can the Po be detected in the olfactory projection. As described in Chapter 4, the dorsal root, which consists of neurite projecting from the DRG to CNS, is not Po positive at E l2 when immunoreactivity of GAP-43 clearly shows that the nerves have reached their CNS target. The delay of Po expression in the neurite of olfactory nerves projecting toward CNS is similar to what was found during development of the DRG. My study of the expression of Po mRNA in the rat olfactory nerves agree with what was reported in the chick by Norgren et al. (1992). The study of Norgren also showed that the Po protein, detected by 1E8 monoclonal antibody, was expressed up to the olfactory nerve layer and not in the glomerular layer.

The periphery of the olfactory epithelium is positive at E l4. The identify of the weak Po positive cells in the periphery is puzzling at first sight. A plausible explanation for this is that the bone structure encapsulating the epithelium is developing and only separated small bundles of the nerves could pass through the cartilage/bone structure. GAP-43 and NCAM immunoreactivity in this area demarcating the olfactory nerves show that fine fibrous nerves are sieved through the lamina cribrosa and converging into a much thicker band of olfactory nerve (Gong and Shipley, 1996). The weak, separated Po positive cells therefore represent the cells located at the exit point on the

191 bone structure. Experiments in which the E14 olfactory epithelium was used for immunoreactivity for low affinity NGF-R showed a similar picture. Scattered NGF-R positive cell on the periphery of the olfactory epithelium were separated from the more peripheral, highly NGF-R immunoreactive nerve, presumably by the NGF-R negative bone structure (Figure 8D). The presence of Po positive cells in the peripheral of the epithelium supports the hypothesis that the olfactory Schwann cells are generated from the precursors in the olfactory epithelium, as studies using electromicroscopy and tissue culture suggested (Marin-Padilla and Amieva, 1989; Chuah and Au, 1991)

5.3.9 The new discovery of Po expression in the adult olfactory nerves suggests a different mechanism of regulation from gut and non myelinated cells Using teased nerve preparation, the Po gene was detected in the adult olfactory nerves at a condition when the adult cervical sympathetic trunk was not positive for the signal. In other word, the Po gene in the adult olfactory nerves, which are largely not myelinated, was not actively inhibited like in the unmyelinated adult cervical sympathetic trunk (CST) and in the gut. As shown in chapter 3 and 4, a basal Po expression is detected in the embryonic CST at E l8 and in the migrating neural crest in the gut at E l3. This basal Po gene expression is down regulated when the CST matures, or when the enteric glia reach its target in the gut. This down regulation is not observed in the olfactory nerves, suggesting that the regulation of Po gene is unique in this system. The difference in Po gene regulation might reflect the difference in maturity rather than a substantial phenotypic difference from other non­ myelinating Schwann cells. Firstly, as demonstrated in Chapter 3, a basal level of Po is expressed in Schwann cell precursors, and it is differentially regulated as the nerves mature in the myelinating and non-myelinating Schwann cells. The Po expression in the olfactory nerves resembles the embryonic expression and is not regulated as it is in adult sciatic nerves (Lee et al., 1997). Moreover, morphologically the axons of adult olfactory nerves are not myelinated and are not ensheathed by connective tissue (DeLorenzo, 1957), an arrangement similar to the developing sciatic nerve associated with the Schwann cell precursor (lessen et al., 1995). When co-cultured, the olfactory Schwann cells can myelinate DRG neurons, suggesting that when provided with

192 further differentiation factors, the olfactory Schwann cells can express the range of genes necessary for myelination (Devon and Doucette, 1992). Lastly, when transplanted to transected CNS tract, the olfactory Schwann cells help the CNS axon to regenerate as well as reenter host pathways, which the embryonic Schwann cells also do better than the adult Schwann cells (Li et al., 1997).

5.3.10 A possible role for Po expression as permissive substrate for axons during early development and regeneration Another interesting observation is that the presence of Po in growing nerves that are actively entering the CNS is also observed in the entry/exit point for the cranial nerves and spinal nerves. As described in chapter 4, a high level of Po expression is found on the border of the neuroepitheium and adjacent mesenchymal tissue where motor axons exit the neural tube in the trunk region, as well as in the entry/exit zone of the cranial nerves. This expression forms a band in the trunk area along the length of cranial-caudal axis and it not interrupted by the absence of neural crest cells in the posterior half of the somite. High Po expression in both trunk and head area coincident with the time of active entering and exiting of the nerves takes place beginning at E l2 rat in part of the embryo. In vitro Po is known to promote neurite outgrowth when coated as a substrate (Schneider-Schaulies et al., 1990). These facts together with the fact that the Po is ever present in the olfactory nerves, which are known for their ease in crossing the CNS/PNS boundary, suggests that Po might play a role in development and could facilitate regeneration by assisting the nerve to cross the CNS/PNS border. Again, this is speculation and needs further research.

193 P o F ^ aDelta-1 t > A

A B

B

#

Po PLP

Figure 5-1 At E ll Po is not detected in the epibranchial placode derived neurons, although it is clearly seen in the otic vesicle and trigeminal ganglion (A), which are already Delta-1 positive at this stage (B, arrows). Arrow heads in A and B point to 7th and 8th ganglion. OV; otic vesicle

Figure 5-2 Po and PLP/DM-20 in the otic vesicle (A) At E12 expression of Po within the vesicle is not uniform. Labeling is more evident in dorsomedial and ventrolateral areas and in the developing endolymphatic duct; (B) At ElO in mouse otic vesicle (equivalent to E l2 rat) lacZdriven by the PLP/DM20 promoter in a transgenic mouse is also seen in dorsomedial and ventro-lateral areas. In both sections the hindbrain lies to the left of the section, and dorsal regions are at the top of the figure. V ) '.y A ed

sc

g c

B

Figure 5-3 A series of horizontal sections through the inner ear at E14. PO labelling is evident in the endolymphatic duct (ed), semicircular canal (sc), vestibule (v), cochlear duct (cd), and acoustic ganglion (g). Each section is 20pM thick, and sections are lOOpM apart. - I -

%» m . , . ' = S

V'

Figure 5-4. A representive section which is more anterior to fig 5-3. The hindbrain lies to the top of the section. PO labelling is evident in the vestibule (v), saccule (s), cochlear duct (cd), nerve roots leading to hindbrain (r), and acoustic ganglion (g). BMP-4

iSC

Po C-ret

Figure 5-5. Comparison of PO labelling with that of (A, B) BMP-4 and (C,D) c-ret. AB and CD are adjacent pairs. The BMP-4 and c-ret sections are 40 |iM apart, and they were 20pM apart from their adjacent Po sections. The expression of BMP-4 and c-ret are punctate but not idential to each other, while the PO is complementary to the two sensory markers, sc: semicircular canal, c: cochlea, g: acoustic ganglion. Figure 5-6. Comparison of PO labelling with that of (A,B) BMP-4 and (C, D) serrate-1 in sections of the cochlea shows that the pattern of distribution of PO cells is clearly complementary to that of BMP-4 and serrate-1. Part of the ganglion (g) is visible to the left of the cochlea in C

Figure 5-7. PO and c-ret labelling of consecutive sections through the vstibule (v) and cochlea (c). (A) PO labelling is widespread but non-uniform; (B) c-ret labelling is highly restricted to small areas in the saccule and cochlea (arrowed).

198 BMP^

Q * t.,

Po

✓ -*•' ^''NkZ^^ -■. " \:'" :''^: ^ r l L ^ c-ret \ V X B AD CST

Figure 5-8 (A) Portion of a whole mount of the olfactory region at El 3. Labelling is seen in the developing olfactory nerve (n), as it projects to the telecephalon (t); (B) in a horizontal section taken at E14 through the olfactory epithelium Po is absent from the olfactory epithelium (e) but is seen in developing nerves (arrow). Po expression persists throught adulthood in the olfactory Schwann cells (D), which is not myelinated, while in the cervical sympathetic trunk Po is only present in myelinating Schwann cells (C). Chapter 6 General Discussion

201 The aims of this study were firstly to study the regulation of the basal level of Po gene expression during development and regeneration of the PNS, and secondly to study this basal level of Po expression during the earliest stages of the formation of the neurogenic placodes and neural crest and its significance. This study confronted the traditional view of Po as a gene that is only expressed by myelinating Schwann cells as a structural protein and adhesion molecule. The expression of Po mRNA itself proved a good marker in studying the differentiation and specification of PNS glial cells from the neural crest. It was also unexpectedly expressed in placodes, which like the neural crest, delaminate from the neural plate/neural tube, and in the notochord, which also separates during early development from the developing neural plate, again providing a molecular marker for studying the origin and differentiation of PNS versus CNS structures during neurulation. The expression in the inner ear may facilitate the study of the nonsensory epithelium of inner ear, parts of which are important in maintaining the ionic balance of the endolymphatic fluid.

The Po gene in the PNS glial lineage is under a ‘three-gear’ regulation. I described a basal level of Po expressed in cells that give rise to all PNS glial cells, including the Schwann cell precursor (described in chapter 3), the immature enteric glial and satellite cells (described in chapter 4), well before myelination. Po is constitutively expressed in the PNS glial lineage well before myelination, and is actively down- regulated in most of the sub-types except myelinating Schwann cells and olfactory Schwann cells (chapter 5). This basal level of Po expression is regulated differently from the high level observed during myelination in that it is constitutively expressed unless actively inhibited, while the high level observed during active myelination is due to a further up-regulation, which is down-regulated to the basal level when axonal signals are removed (chapter 3). The high level seen during myelination is not due to a release from an inhibition before myelination, as the basal level seen in olfactory nerves does not automatically lead to myelination even though no inhibition is present. In adult non-myelinating cells, where basal Po is actively inhibited, transection results in disinhibition and return to basal Po mRNA levels (chapter 3).

202 The significance of this basal Po expression is intriguing. Based on its adhesive properties, it is reasonable to propose that Po might play a role during migration and organogenesis as an adhesive agent. A critical feature of the adhesive molecules utilized for this purpose is that the affinity cannot be too high, since it is useful for the precursors of PNS glial cells to travel to their final position without ‘getting stuck’ halfway through migration. Although the early expression of Po protein is not shown in vivo in my study, using a more sensitive assay Po protein is shown to be expressed ubiquitously in Schwann cell precursors in vitro, and can be shown in neural crest cell cultures (Lee et al., 1997). During development the low level of Po could serve as a good adhesive agent during migration, as the adhesion of an adhesion molecule is regulated by the density of the molecule on a particular surface (for example, D’Urso et al., 1990). Furthermore, the mechanism of regulating the basal level must be compatible with that for cell proliferation. Indeed my observations showed that the basal level is detected at a time when the Schwann cell precursors or immature Schwann cells are actively dividing (Stewart et al., 1993; lessen et al., 1994) . Subsequently when the Schwann cell precursor or immature Schwann cells reach their target, theoretically this adhesiveness is no longer needed and was hence actively inhibited, or in the case of myelinating Schwann cells, upon cessation of proliferation, further up-regulated.

As myelin is a relatively new invention during evolution, constitutively expressed Po in different sub-types of PNS glia when not subjected to inhibitory signals, while high levels are only observed in myelinating Schwann cells, suggests that Po, at some point through evolution, may have been reinvented by the vertebrates from a mild adhesive molecule to a compaction reagent in myelination. This hypothesis is supported by 3 observations: 1. the composition of the N-linked oligosaccharide of the extracellular domain of Po is developmentally regulated (Brunden, 1992). 2. Data from the crystal structure of Po indicates that the interaction between the extra­ cellular domain, which is believed to contribute to the adhesive ability between the juxtaposing membranes, is weak. The adhesive interaction only becomes strong when the level of Po expression level is high (Shapiro et al., 1995). 3. The early Po expression precedes that of Krox-20, which is believed to be important during

203 myelination (Chapter 4). Therefore basal Po expression is probably regulated differently from the myelinating level and has another role during migration of the crest cells and precursors of PNS glia. A further examination of Po-like molecules in invertebrates, starting from the more primitive species of chordate, for example, the tadpole larva of ascidian tunicate, or amphioxus, would give a hint as to whether Po has been derived from a weak adhesive reagent to play major role during myelination. If this hypothesis holds true, there should exist some species that do not have myelinated nerves but have Po-like protein in their glial cells.

Apart from the migrating immature glial cells that are destined to form sub-types of PNS glia cells as described above, a basal expression is also found in cells of the ventral exit zone (chapter 4), olfactory glia cells (chapter 5), and Schwann cells after axotomy (chapter 3). These cells are known for their ability in permitting the passage of neurites from a PNS environment to CNS environment, or vice versa, during regeneration. In view of the fact that Po is known for its role in promoting axonal contact and neurite outgrowth (Schneider-Schaulies et al., 1990), and that members of the Ig superfamily expressed in Schwann cells (LI, MAG, Po) are known to have a role in initiation of axonal ensheathment by Schwann cells (Doyle and Colman, 1993), this property of ventral exit zone cells and olfactory glia is not so surprising. It would be interesting to investigate whether the continued expression of Po by olfactory ensheathing cells and the relatively high level of protein expression in dorsal root (and ventral root) entry zone cells (Golding et al., 1997) is directly related to their ability to permit axon outgrowth.

Besides derivatives of the neural crest, Po is expressed in the ventral exit zone that is probably neural tube derived (chapter 4), the notochord (chapter 4) and placode derived structures (chapter 5). The structures that express Po share a common feature; they are part of the neural plate border and are generated between the neural plate and the surrounding non-neural mesoderm during development. The location of the anlage of these ‘quasi-PNS’ structures (ventral exit zone cells, notochord, placodes) and that of neural tube are continuous, and the difference from the neural tube is that they are excluded from the future CNS during development. As mentioned above, Po

204 is inhibited in any environment that is similar to CNS (i.e., the enteric ganglia). Is exclusion from neural tube and hence CNS a prerequisite of Po expression in neural plate derived structures? Or does expression of Po define the difference between the CNS and PNS environment? In my study I found that PLP is expressed in placode derived structures, notochord, and has additionally been reported in precursors of CNS glia (Timsit et al., 1992, 1995). The fact that two major myelin proteins are expressed in ‘quasi-PNS’ structures, as mentioned above, suggest a lineage relationship of the neural crest and its derivatives and these ‘quasi-PNS’ structures and their derivatives.

In chapter 4, I demonstrated that Po is expressed in a sub-population of cells of the neural crest which take the ventral-medial pathway, and that it is only expressed when they move more ventrally and are therefore probably more mature. It is restricted to the anterior half of the somite, and is not in the melanocytes which take the dorsal- lateral pathway. It is expressed in crest cultures only after 2 days, after the time that the glial and neuronal lineages separate, and it is the earliest marker characterized so far for PNS glia. Therefore it is expressed in the glial lineage, and only after the segregation of glial and neuronal lineages. These findings have contributed substantially to the study of the Schwann cell lineage, especially at a stage when its influence on immature neurons are crucial for the survival of the latter. This also throws light on the paradox of the multipotentiality versus restricted fates of the neural crest derivatives. In the past the study of neural crest development has suffered from an artificial in vitro culture system and neglect of cell types other than neuronal and melanocytes among the derivatives. With an improved culture system and a marker that specifies the early stages of the glial lineage, in my study it is shown that the fate restricted, single potential neuronal or glial precursors appear in larger quantity and earlier than shown in other culture systems.

In chapter 5 I demonstrated that Po is expressed in the non-sensory epithelium of the inner ear, based on a comparative study using several markers that are known to mark the sensory area of the embryonic inner ear. The expression patterns of BMP-4, c-ret and serrate-1 were compared to that of Po, and the result showed that the pattern of

205 Po is complementary to that of the markers of sensory epithelium, at least in the cochlear region. Two possible roles of Po expression during early development of inner ear are proposed, one being that Po acts as an adhesion molecule during organogenesis, and the other being that Po might be expressed in the less well known ‘fibrocytes’ in the stria vascularis (or tegmentum vasculosum in chick). Several adhesion molecules have been described in the otic vesicle and during development of the inner ear (Richardson et al, 1987; Meyer Zum Gottesberge and Mai, 1997; Legan and Richardson, 1997), and although not directly compared in my study, their expression patterns described in the literature are not the same as Po. As Fekete’s boundary theory proposed (1996), boundaries that are important in establishing future morphological or functional territories of the inner ear, could be derived in a number of different ways from variations in the expression of a number of different molecules which can be cell surface molecules or diffusible signalling molecules. Boundaries formed by patches of adhesion molecule expression are especially interesting when thinking about the possible function of Po Adhesion molecules are possibly physically involved in modelling the shape of the inner ear, as opposed to the up­ stream homeobox genes whose expression pattern is probably not closely associated with structures moulded during morphogenesis since the signal is passed down via a cascade of molecules. A map of expression patterns of adhesion molecules would probably predict pairs of adhesion molecules that act via heterophilic binding during organogenesis. A comparison of this map to the ‘hotspots’ of apoptosis might give pointers to the mechanism underlying generation of the shape of inner ear. One argument against this theory is that there is no report about malformation of the inner ear in Po knock-out mice (Giese et al., 1992). Therefore alternatively, instead of being functional, early Po in the ear could be a ‘fossil’ trait that has been left over during evolution. However the fact that PLP is also expressed in the otic vesicle makes it less possible that two highly versatile genes which are known to play major roles elsewhere are expressed during the generation of the inner ear simply for nothing. Anyhow it is the earliest marker for cells in the non-sensory area, where many defects in hearing are rooted. Considering the whole battery of cells devoted to pumping potassium ions back to the endolymphatic fluid, it is surprising that to date very few hearing impairments have been ascribed to mutation of these cells, probably

206 because of undue attention to melanocytes. Recent success in culturing these highly specialized ‘fibrocytes’ will enable the study of these cells in vitro and more precision in describing their phenotypes. The next few steps would be, firstly a detailed description of Po and its correlation to more differentiated cells only found at later stages of the development of the inner ear in the non-sensory area, and secondly how Po is regulated and expressed in these cells. From my study it could be concluded that Po is not regulated in the same way as in PNS glia, since the very high level of Po is found in E l4 rat inner ear in an area that lacks innervation.

207 Chapter 7 References

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