The Function of the Hyalectan Class of Proteoglycans and Their Binding Partners During Vertebrate Development

No part of this digital document may be reproduced, stored in a retrieval system or transmitted commercially in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Chapter II

The Function of the Hyalectan Class of Proteoglycans and Their Binding Partners during Vertebrate Development

Frédéric Brunet,1 ,* Christopher Kintakas,2 Adam D. Smith2 and Daniel R. McCulloch2,† 1Institut de Génomique Fonctionnelle de Lyon, UMR5242 CNRS/INRA/Université Claude Bernard Lyon I/ENS, Ecole Normale Supérieure de Lyon, Lyon, France, 2Molecular and Medical Research (MMR) Strategic Research Centre, Molecular Medicine Research Facility, School of Medicine, Faculty of Health, Deakin University, Australia

Abstract

The hyalectan family of extracellular matrix (ECM) chondroitin sulphate proteoglycans comprises aggrecan, brevican, neurocan and versican. Although they share common structural domains including a G1 domain that is invariably linked to hyaluronan, their tissue distribution and biological roles in vertebrates are diverse. During vertebrate embryonic development, complex interactions between cells and their surrounding ECM precede profound cellular behaviour and morphogenetic events. Studies in several vertebrate species have identified specific roles for the hyalectans in numerous embryonic processes from early through late embryogenesis that are necessary for shaping the body plan and subsequent morphogenesis. Of particular note is the wide distribution of versican throughout the vertebrate embryo and its proteolytic processing by the proteoglycanase family of disintegrin-metalloproteinases (ADAMTS). Versican is differentially spliced into four distinct isoforms: V0, V1, V2 and V3, with overlapping and specific tissue distribution within the vertebrate embryo. V0 and V1 are the most

* [email protected]. † Corresponding author: Dr Daniel R. McCulloch, Phone: +61-3-52272838, Facsimile: +61-3-52272615, Email: [email protected]. 50 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

widely distributed isoforms, and also those that contain a specific ADAMTS proteoglycanase processing sites. Studies in the chicken (Gallus gallus) and mouse (Mus musculus) have revealed specific roles for versican during embryogenesis including cell migration, apoptosis, morphogenesis and organogenesis, whilst few studies have identified roles for versican in the zebrafish embryo (Danio rerio). Herein we review the major roles identified for the hyalectans during vertebrate development with a general focus on versican. We further provide new insights into the conservation and diversity of versican and other hyalectans, and their hyaluronan binding proteins in the developing zebrafish embryo along with their potential processing by ADAMTS proteoglycanases. We identify the developing zebrafish embryo as an ideal platform to enhance our understanding of hyalectan biology throughout vertebrate embryogenesis.

Introduction

The Hyalectan / Lectican Class of Chondroitin Sulphate Proteoglycans

The extracellular matrix (ECM) is a dynamic environment comprising several classes of macromolecules, which confer not only structural integrity but also bioavailability of growth factors and cytokines to their surrounding cells’ and tissues’ microenvironment. Proteoglycans are specialised ECM macromolecules that consist of a core protein with large glycosidic side chains called glycosaminoglycans (GAGs) commonly covalently attached to serine residues that occur within intermitted repeats of serine-glycine (or glycine-serine) GAG motifs. GAGs can be further modified by the addition of covalently attached chondroitin sulphate (CS), comprising repeating D-glucuronic acid (GlcA) and N-acetyl-D- galactosamine (GalNAc) residues, dermatan sulphate (DS) whereby the GlcA residues are epimerized into L-iduronic acid (IdoA), heparan sulphate (HS), which comprises glucuronic acid (GlcA) linked to N-acetyl glucosamine (GlcNAc) and keratan sulphate (KS), which comprises linear polymers of lactosamine, 3Galβ1–4GlcNAcβ1 moieties [1]. The diverse modifications of GAGs with respective sulphate moieties give differential biological properties to each macromolecule that includes substrate binding and ligand specificity, osmotic potential for tissue rigidity, and growth factor binding properties. Proteoglycans are broadly classed according to their GAG composition, for example the heparan sulphate proteoglycans (HSPGs) are modified by heparin/heparan sulphate and the chondroitin sulphate proteoglycans (CSPGs) are modified by chondroitin sulphate. However, a much greater diversity exists depending upon their core protein properties, for example, the small leucine rich proteoglycans (SLRPs), as well as other proteoglycan-specific binding properties. Among the many classes of proteoglycans are the hyalectans (or lecticans), which have emerged as important mediators in a broad range of biological processes at the cellular and tissue organisational level. The hyalectans were named after their ability to bind to hyaluronan (HA), a large non-proteinaceous, non-sulphated GAG and their additional lectin- binding properties [2]. Lectins are a diverse class of sugar-binding molecules whose ligands represent specific carbohydrate moieties. The most well known function of lectins is their ability to clump red blood cells (hemagglutinin) however they have proven to be powerful tools in dissecting roles for carbohydrate biology in vertebrates. The hyalectans specifically comprise the proteoglycans: aggrecan, brevican, neurocan and versican (Figure 1), and are The Function of the Hyalectan Class of Proteoglycans … 51 classically CSPGs, although they may either be modified by other classes of GAGs, or some splice variants may be devoid of GAG attachment motifs.

Figure 1. The hyalectan/lectican class of proteoglycans. KS = keratan sulphate, CS = chondroitin sulphate, HABR = Hyaluronic Acid Binding Region, G3 domain: EGF-like = epidermal growth factor- like, CRP-like = complement-regulatory protein-like.

Hyaluronan and Hyaluronic Acid Synthases

Hyaluronan (HA) is a large, predominantly extracellular/pericellular-localised non- sulphated GAG comprising N-acetylglucosamine and glucuronic acid disaccharide repeats [(1-3)-β-d-GlcNAc-(1-4)-β-d-GlcA-]. HA is synthesised by hyaluronic acid synthase (HAS), of which there are four identified in vertebrates and three in tetrapods (HAS1, HAS2 and HAS3) [3]. HA exists in medium to high molecular weight species (up to 4-5 x 106 Daltons) and is widely distributed throughout the vertebrate embryo and in adult tissues. Among the many described properties of HA is its hydrophilic potential, drawing water into the extracellular environment to provide it with lubricant and viscosity. Cell surface receptors for HA include the widely expressed and well characterised CD44 glycoprotein (Figure 1) as well as receptor for hyaluronic acid mediated mobility (RHAMM), and others that are able to regulate cell signalling events through HA-stimulated binding. An additional important feature of HA is its capacity to bind the hyalectan class of proteoglycans: aggrecan, brevican, neurocan and versican, along with the link proteins (Figure 1 and described in this chapter 52 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. forthwith). Large aggregates of HA-bound hyalectans (and link proteins) provide tissue with a rigid structure and allow resistance and strain to be placed upon tissues such as cartilage and tendon without adverse consequences.

Hyaluronan and Proteoglycan Link Proteins

The hyaluronan and proteoglycan link proteins (HAPLNs) comprise four evolutionary conserved mammalian genes (HAPLN1, HAPLN2, HAPLN3 and HAPLN4) with differential expression patterns in vertebrates. HAPLN2 and HAPLN4 expression is predominantly restricted to the central nervous system (CNS) and their protein products bind to brevican and neurocan respectively, while HAPLN3, whose protein product binds to aggrecan, is widely expressed [4]. HAPLN1, also known as cartilage link protein-1 (CRTL1) binds to both versican [4] and aggrecan [5] and functions to stabilise aggrecan monomers to HA in cartilage, but is also expressed in other vertebrate tissues as described forthwith in this chapter.

Aggrecan

Aggrecan (a large aggregating proteoglycan) is the predominant proteoglycan found in cartilage. The importance of aggrecan for normal vertebrate development and growth is exemplified by heritable defects in the aggrecan gene causing osteochondritis (OMIM: 165800), spondyloepimetaphyseal dysplasia, aggrecan type (OMIM: 612813), and spondyloepiphyseal dysplasia, Kimberley type (OMIM: 608361). Both KS and CS moieties are found attached to the aggrecan core protein (Figure 1) whereby KS in cartilage can be linked to serine or threonine residues [1] although the distribution pattern of GAG modifications are variable across vertebrates and tissues within species. Aggrecan is further diversified by alternative splicing, leading to alterations in the binding properties of its G3 domain, which contains a C-type lectin and epidermal growth factor (EGF) motif (Figure 1) and collectively binds fibulin-1 and -2 and tenascin-C and -R [6]. Aggregates of aggrecan are created by several aggrecan molecules binding to HA, mediated by HAPLN1 creating a bottle-brush like appearance. The highly negatively charged CS moieties have significant osmotic potential and attract water into the local tissue environment. Hence, tissues rich in aggrecan are generally rigid in structure and can resist compression and bear load; both necessary properties of articular cartilage but also tendon and, in some vertebrates, heart valves. Aggrecan can also be found in tissues such as the adult brain, eye, spinal cord, and transiently in embryonic structures.

Brevican and Neurocan – The CNS Specific Hyalectans

Brevican (a brain-specific proteoglycan) is a hyalectan predominantly expressed in the brain and CNS. Brevican has attracted significant attention because of its ability to mediate neurogenesis and its involvement in the pathology of glioma [7]. Neurocan (a neuron-specific proteoglycan) is also predominately found in the adult CNS but is widely distributed The Function of the Hyalectan Class of Proteoglycans … 53 throughout vertebrate development before becoming CNS confined. Together, brevican and neurocan represent the CNS-specific hyalectans, and as discussed below, have roles in neurogenesis, existing in complex with other ECM molecules in perineuronal nets (PNNs) of the brain, contributing to neuronal differentiation, extension and glial cell migration.

Versican

Versican (a versatile proteoglycan) is perhaps the most widely distributed proteoglycan in the developing vertebrate embryo and adult tissues. The G1 domain of versican can bind HA in a similar manner to that of aggrecan (and indeed all of the other hyalectans), mediated by HAPLN1. Versican exists as four distinct splice variants designated V0, V1, V2 and V3. Differing in structure, the V0 (full-length) splice variant contains both the GAG-alpha and GAG-beta domain; the V1 variant contains only the GAG-beta domain, the V2 splice variant contains only the GAG-alpha domain and V3, not technically a proteoglycan, does not contain GAG attachments on its core protein (Figure 2). In the vertebrate embryo and adult, V0 and V1 are the most widely distributed forms of versican, present for example in the mouse brain, heart, lung, spleen, skeletal muscle, skin, tail, kidney, and testis [8], whereas the V2 and V3 versican species are generally restricted to the CNS, although these forms can be found elsewhere albeit transiently during embryogenesis and at lower levels in adult tissues. Versican is found particularly highly expressed in mouse embryos at embryonic days E13, E14 and E18 [8].

Figure 2. Versican splice variants. CS = chondroitin sulphate, HABR = Hyaluronic Acid Binding Region, G3 domain: EGF-like = epidermal growth factor-like, CRP-like = complement-regulatory protein-like. 54 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 3. The mammalian ADAMTS proteoglycanases. SS = signal sequence, Pro = pro-domain, Cat = catalytic domain, Dis = disintegrin-like domain, TS-1 = thrombospondin type-1 repeat, Spac = spacer domain, N-Gly = N-glycosylation, C-mann = C-mannosylation, O-fuc = O-fucosylation. Residue numbers are defined by the following consensus sequences, where the underlined residue is numbered: Furin cleavage site: RXXR, N-glycosylation: NXS/T, C-mannosylation: WXXW (multiple tryptophan residues are predicted to be mannosylated within any given consensus sequence), O-fucosylation: CXXT/SCXXG. Asterisks denote a C-mannosylation site shortly followed by an O-fucosylation site, i.e.: WXXCXXT/SCXXG. Amino acid numbers correspond to human ADAMTS proteoglycanase annotation.

The A Disintegrin-Like and Metalloproteinase Domain with Thrombospondin-1 Repeats (ADAMTS) Proteoglycanase Family

The ADAMTS family of zinc-dependent metalloproteinases comprises 19 mammalian extracellular secreted enzymes that are divided into clades based upon their substrate specificity and evolutionary conservation [9]. Together with the more recently discovered ADAMTS-like (ADAMTS-L) family, ADAMTS enzymes have recently taken the spotlight in ECM remodelling during development and disease. The so-called ADAMTS proteoglycanases (ADAMTS-PGases) are an intriguing evolutionary-conserved clade of the ADAMTS family with substrate specificity directed towards the remodelling of the hyalectans, although ADAMTS-PGases can also direct their proteolytic activity towards non- hyalectan ECM substrates such as decorin and biglycan (both SLRPs), and non-proteoglycan substrates such as fibronectin [10]. ADAMTS-PGases comprise ADAMTS1, -4, -5, -8, -9, 15 and -20 [11] (Figure 3), although ADAMTS16 and ADAMTS18 have also been shown to have PGase activity directed towards aggrecan [12], albeit at lower levels. Together, the ADAMTS-PGases are represented in a diverse array of tissues in both the developing vertebrate embryo and adult where they demonstrate either solitary or overlapping functions in so-called co-operative proteolytic networks. Although the hyalectans can be processed by proteases other than ADAMTS-PGases, the best example being the matrix metalloproteinases (MMPs), hyalectan remodelling by ADAMTS-PGases has emerged as an important process in both physiology and disease. The fundamental process of substrate cleavage by ADAMTS-PGases has profound effects upon embryonic morphogenesis throughout diverse sites of the developing embryo and equally The Function of the Hyalectan Class of Proteoglycans … 55 profound destructive properties in disease, particularly in arthritis [13-14]. All hyalectans have sites of ADAMTS-PGase proteolysis, with the most conserved generally being in relatively close proximity to their G1 N-terminus [7, 15-17].

Hyalectans in Vertebrate Development

Embryogenesis is a Conserved Set of Mechanisms amongst Vertebrates

Embryonic development is the complex process whereby, in mammals, two gametes’ pro-nuclei fuse after fertilisation within a single cell called the zygote, which develops into a multi-cellular, multi-functional organism. The highly conserved mechanisms underlying vertebrate embryogenesis represent a powerful analytical tool for developmental biologists to dissect embryonic processes in order to learn concepts broadly applicable across vertebrate species. These processes are commonly evaluated in several mammalian, avian, amphibian and fish vertebrate models that include the frog (Xenopus laevis), the chicken (Gallus gallus), the quail (Coturnix japonica) the rodents: mouse (Mus musculus) and rat (Rattus norvegius), and more recently the teleosts zebrafish (Danio rerio). One collective advantage of those models is their ease of manipulation at the genetic level. Higher order vertebrates such as the pig (Sus scrofa), the dog (Canis familiaris), non-human primates such as the rhesus monkey (Macaca mulatta) for example and the human primates (Homo sapiens) are also readily utilised in research where possible, although genetic manipulation for the purposes of discovering the function(s) of one or several genes is less common due to their complexity and ethical constraints. Although the gestational periods vary widely between vertebrate developmental models many important embryonic processes are well described and indeed tightly conserved, and can thus be extrapolated across species and exploited accordingly to discover biological processes alike. The consequences of hyalectan synthesis and turnover, i.e. proteolysis, during embryonic development are discussed forthwith in this chapter, in context of the expression and roles of each hyalectan during key developmental processes.

Fertilisation, the Formation of the Blastocysts and Embryonic Stem Cells

The early development of the vertebrate embryo has a common goal: to form a 3- dimensional body plan. In mammals, after pro-nuclei fusion, the process in which the female and male haploid (1N) gametes’ genetic material, i.e. their DNA fuse to form a diploid cell (2N) with a full complement of chromosomes, the large 2N zygote undergoes a series of cleavages that create a fluid-filled multi-cellular structure called the morula (ancient Greek for mulberry). This morula then undergoes cellular repositioning to form the blastocyst (ancient Greek for germ burgeon), where an inner cell mass (ICM) can be clearly discerned; the embryo is now called the blastula. The ICM represents the embryo proper, i.e. all cells of the ICM will form embryonic structures. The ICM is surrounded by amniotic and chorionic cavities within an outer supporting structure called the chorion. Cells of the ICM are totipotent, or at least, pluripotent and indeed are the rich source of embryonic stem cells (ESCs), that have been subsequently cultured in vitro, creating powerful tools to study stem cell differentiation; the grass-roots of tissue engineering. 56 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Hyalectans and ESCs

When human ESCs (hESCs) are differentiated to form cardiomyocytes, there is an early accumulation of HA and versican. The versican species synthesised by hESCs contain more N- and O-linked sugars than that found in subsequently differentiated cardiomyocytes. Although the expression of all splice variants of versican (Figure 2) increase upon hESC differentiation, V1 is the predominant form [18]. HA species also differs throughout hESC differentiation whereby high molecular weight HA is predominant in differentiated cardiomyocytes while lower molecular weight species are predominant in hESCs. This change is accompanied by the absence of HAS3 in cardiomyocyte cultures and its corresponding presence in hESCs. When mouse ESCs (mESCs) are differentiated into embryoid bodies an epitheliomesenchymal transition (EMT) occurs. Similar to hESCs, the full complement of Has genes and versican splice variants are expressed in mESCs, and Has2 and versican V0 and V1 splice variants’ mRNA become the predominant species throughout differentiation. A 70 kDa versican immunoreactive species, corresponding to the G1- DPEAAE fragment generated by ADAMTS-PGase proteolysis [16] is also strongly associated with mESC differentiation as is co-expression of both Adamts1 and Adamts5 alongside Has2. The epithelial cells (E-cadherin positive cells) of the embryoid bodies exclude versican, whilst mesenchymal cells (N-cadherin positive cells) are versican enriched [19].

Gastrulation and Neurulation

In mammals, after the formation of the blastocyst, the ICM is reorganised into two germ layers, the bilaminar embryo or germ disc. The dorsal side is called the epiblast, which is the primitive ectoderm, whilst the ventral side comprises the hypoblast or primitive endoderm. Specific to mammals, birds and reptiles is the first sign of bilateral symmetry that appears as a condensation of cells dorso-medially, i.e. through the midline on the dorsal primitive ectoderm, and is called the primitive streak. The cranial aspect of the primitive streak is indicated by the primitive node, also known as Henson’s node in the chicken, which is responsible for organising left-right asymmetry through the expression of transforming growth factor-β (TGF-β) family members such as NODAL and ACTIVIN. The fish is more clearly defined early in development by the identification of the two distinct poles: the animal pole, where cells of the embryo per se are located and the vegetal pole, where the yolk cell is located. At the animal pole, the blastoderm comprises a surface enveloping layer, which engulfs the deep layer that in-turn arrests on the yolk syncytial layer towards the vegetal pole. At the dorsal end of the animal pole in the deep layer, cells begin to ingress and form a distinct hypoblast cell layer. The deep layer that now comprises the epiblast and hypoblast, the fish equivalent to the bilaminar germ disc, is called the shield. In all vertebrate embryos, once the bilaminar germ disc is formed, cells of the epiblast undergo an EMT that allows them to break cell adhesion with their neighbouring cells through a loss of expression of E-cadherin, and then elongate and migrate by reorganisation of their cytoskeleton. In mammals, birds and reptiles, the cells migrate through the primitive streak and node whilst in the fish, the dorsal vegetal cells define the Nieuwkoop-center-like region [20-21] and later the Spemann organizer, which is induced during mid-gastrulation The Function of the Hyalectan Class of Proteoglycans … 57 from signals arising from the Nieuwkoop-center-like region, dorsalising the mesoderm, [22- 23]. In amphibians, the Nieuwkoop center mediates convergent-extension cell movements in both mesoderm and ectoderm [24-25]. This is the beginning of gastrulation and the mammalian embryo is now known as the gastrula whilst in fish it is referred to as epiboly. Gastrulation is the key process that converts the bilaminar germ disc into the trilaminar germ disc comprising the three fundamental germ layers of the body plan: the dorsal ectoderm, the internal mesoderm and the ventral endoderm.

Hyalectans and Gastrulation and Neurulation

Global hyalectan knockout mouse models that independently represent aggrecan, brevican, neurocan and versican gene inactivation indeed escape gastrulation defects. So too does the aggrecan “knock-in” mouse whose ADAMTS-PGases cleavage site within the interglobular (G1-G2) domain (IGD), the Glu373↓Ala374 site, is uncleavable [26], suggesting neither aggrecan nor its cleavage at the major site of proteolysis by ADAMTS- PGases is an absolute requirement for early developmental processes. On the other hand, the Adamts9-/- mouse embryo dies at E8.5, concurrent with gastrulation. Although yet uncharacterised, ADAMTS9 activity towards the hyalectans, and perhaps versican, may have a pivotal role in this process. In the frog, versican is maternally expressed, i.e. its mRNA can be found prior to the onset of zygotic gene expression, whilst zygotic expressed versican mRNA is indeed found at the time of gastrulation [27]. The Has2 knockout mouse dies at E9.5 to E10, also escaping gastrulation defects [28]. However, morpholino oligonucleotide silencing of the zebrafish has2 gene has facilitated the discovery of the role of HA in early development. Has2 morphants display defective migratory patterns during gastrulation, as well as during somitogenesis and primordial germ cell migration (both described forthwith in this chapter) [29]. During gastrulation in the zebrafish, cells converge dorsally to thicken the embryo, a process that is blocked upon has2 silencing and is dependent upon signalling through the activation of the well characterised small GTP binding protein Rac1. HA and its interaction with its receptors CD44 and RHAMM is a potent mediator of cell migration during key embryonic processes, and is particularly noteworthy in the process of cancer cell metastasis [30] where cancer cells migrate from their primary site of colonisation to form secondary tumours throughout the body. Following the gastrula stage, the embryo is now ready to be folded into a three- dimensional structure called the neurula (ancient Greek neuron or fibre). In mammalian vertebrates, bilateral body folding converts the flat trilaminar disc into a tube like structure in a process called neurulation, where the first rudiments of the neural tube, the primordial central nervous system (CNS), can be discerned. In the fish, the neural tube is thought to arise through cavitations. An important process of neurulation in all vertebrates is the formation of the neural crest from neuroectoderm, which is located dorsal to the neural tube and ventral to the overlying ectoderm. Versican expression in the frog continues through neurulation [27] whilst HA is found between the germ layers and is enriched in mesenchymal tissue and in the lumen of the neural tube [31]. Underlying the neural tube is a structure that arises from the axial mesoderm that marks the anterior-posterior (A-P) medial axis of the vertebrate embryo called the notochord. The 58 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. notochord is integral in maintaining body axis symmetry and dictates many early morphogenic processes that give rise to the axial skeleton as well as limb and trunk musculature. The zebrafish has an additional structure that is positioned ventrally adjacent to the notochord called the hypochord. The paraxial mesoderm is positioned dorso-medio-lateral to the notochord, bilaterally, and is differentiated into structures called somites (“whorls” in mesoderm), predominately under influence of notochordal signalling by genes such as sonic hedgehog (Shh). Somitogenesis, like most embryonic processes, occurs cranial to caudal in all vertebrates and most somites caudal to hindbrain will differentiate into structures called sclerotomes, whose superior halves fuse with the inferior halves of their neighbouring sclerotome, ultimately forming the vertebral arches that overlay the neural tube and prospective spinal cord. Somites also differentiate into the dermomyotome, which further differentiates into the dermatome and myotome, giving rise to the dermis, and voluntary muscles of the trunk and limbs, respectively.

Hyalectans and the Notochord

The notochord has been identified as a key area of hyalectan expression whereby an ECM-rich notochordal sheath and ECM-rich adjacent mesenchyme, in some species, consist not only of aggrecan and versican but also collagen II and the HSPG perlecan. The chicken has played a seminal role in the identification of notochordal associated aggrecan. Chicken aggrecan (PG-H) is expressed at stage 16 until day 5, a period which coincides with neural crest cell (NCC) migration and sclerotome differentiation [32]. At least one function of notochordal aggrecan has been elucidated: when added to growth medium, aggrecan induces dorsal root ganglia (DRG) collapse and inhibits DRG axonal growth suggesting its involvement in notochordal-derived repulsion for DRG axons, a process also described in the mouse [33]. Chicken notochordal aggrecan is devoid of KS and has a specific epitope designated as HNK-1, which is not present in chicken cartilage aggrecan [34] indicating differential remodelling between tissues. Chicken versican (PG-M) expression, on the other hand, is noted in mesenchymal rich tissue adjacent to the notochord and neural tube [35] indicating a spatially distinct role for versican as compared with that of aggrecan. The ECM surrounding the notochord of the combinatorial Sox5; Sox6 knock-out mouse is absent, and the notochord undergoes progressive apoptosis [36] exemplifying the necessity for a fully- functional ECM, under the control of Sox transcription factors, for notochordal cell survival. In the human notochord (Carnegie stages 12 and 14) the axial mesenchyme is indeed rich in sulphated GAGs and HA but aggrecan and keratan sulphate are absent, highlighting the possibility that another hyalectan may be predominant in the humans. In the frog, brevican is first observed in the notochord of the neurula [37] while in the zebrafish, hapln1 is expressed in the hypochord and in the sclerotome as well as adaxial cells, which are located adjacent to the notochord bilaterally and fated to become slow muscle fibres [38].

Body Folding and the Ectoderm

Simultaneous mechanisms dictate the cranial and caudal folding of the embryo that is rolled up into a tube following neurulation. Arising from these collective folding events is the The Function of the Hyalectan Class of Proteoglycans … 59 primitive gut tube, which is segmented into foregut, midgut and hindgut structures, and the formation of the intraembryonic coelom, the prospective peritoneal cavity. Mesodermal cavitations at the cranial end of the embryo form the pleuropericardial cavity, which is ultimately segmented by the intrusion of mesoderm to separate the pericardial cavity that encompasses the heart, and the pleural cavity, in which the lungs develop (in land vertebrates). Thus, after neurulation and body folding, a three-dimensional embryonic structure comprising the neural tube, the prospective CNS, the gut tube, the prospective gastrointestinal tract and lungs, the body cavities, and mesodermal anlage of organs such as the heart, is apparent.

Hyalectans and Skin

The overlying (surface) ectoderm of the embryo becomes the epidermis of the skin. In the chicken, Hapln1 (Crtl1) is expressed in ectoderm lateral of the neural plate [39]. Human fetal skin has higher versican content than that of adult skin and in late fetal development versican is restricted to the upper half layer of dermis with the exception of hair follicles and vasculature [40]. In both mouse and human skin, versican is rich at the induction of hair morphogenesis in the dermal papilla anagen phase and diminishes as the follicle progresses through the catagen phase while it is not detectable in the telogen phase [41]. The necessity for versican in the formation of human hair follicles is demonstrated by lentiviral-mediated RNAi suppression of versican, which inhibits aggregative growth of dermal papilla cells [42].

The Neural Crest and Neural Crest Stem Cells

The neural crest, dorsal to the neural tube and ventral to the overlying ectoderm, comprises vertebrate-specific NCCs, which are multipotent and Sox10/p75NTR double- positive expressing cells [43]. NCCs undergo an EMT enabling them to migrate away from their neural crest origin throughout the embryo to form a diverse array of specialised structures including melanoblasts (and other pigment producing cells), sympathetic chain ganglia and neurons of the adrenal medulla, dorsal root ganglia, cranial nerve ganglia, and cranial cartilage and bone anlage [44]. NCCs are also necessary for the normal development of the heart, including aortic-pulmonary septation [45] and structures of the outflow tract, as well for providing inductive signals to instruct the correct development of the pace-making conducting system [46-47].

Hyalectans and NCCs

The V0 and V1 splice variants of versican are inhibitory to NCC migration at low concentrations and it remains questionable whether their inhibitory properties are haptotactic in nature. The inhibitory effect exerted by V0 and V1 versican acts synergistically with fibronectin [43, 48]. As described previously in this chapter, both V0 and V1 versican, and fibronectin are substrates for ADAMTS5, an ADAMTS-PGase whose expression invariably overlaps with V0 and V1 versican during mouse embryogenesis and in adult organs [49] 60 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. suggesting ADAMTS5 may have a role in NCC migration. V0 or V1 versican strongly inhibits the migration of NCCs on a fibronectin substrate by interfering with cell-substrate adhesion [43] and the core versican protein, rather than the GAGs attached to V0 (GAG-alpha and GAG-beta – Figure 2) and V1 (GAG-beta – Figure 2) versican is thought to be involved in exerting the inhibitory effects [43]. In the absence of the ADAMTS-PGase ADAMTS20, i.e. in the case of the homozygous Adamts20 knockout mouse “belted” (bt/bt), NCCs fated to become melanoblasts effectively migrate to their site of colonisation to the dermis, but undergo apoptosis at E13.5, a phenotype exacerbated by Adamts9 haploinsufficiency (Adamts9+/-;bt/bt). The site of melanoblast colonisation is rich in versican, however its corresponding proteolytic processing by ADAMTS-PGases is largely absent in bt/bt and Adamts9+/-;bt/bt mouse embryos [50]. V0 and V1 versican are the prevalent isoforms in the chicken at initial and advanced phases of NCC movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. PG-M/versican (specifically, V0 and V1 splice variants) is transcribed by tissues flanking NCC migratory pathways, as well as by tissues delimiting non-permissive NCC migratory areas. Aggrecan, on the other hand, shows a complementary distribution to PG-M/versican splice variants [51]. In the frog, versican is also expressed in NCC migratory patterns accordingly as well as at the site of NCC arrest [27]. In the Pax3 mutant mouse (splotch), NCC defects are predominant and include pigmentation abnormalities, absent DRG and failure of heart septation. Versican is profoundly over-expressed in homozygous Pax3 mutants and generally not expressed in Pax3 positive cells during NCC migration, suggesting Pax3 may negatively regulate versican [52]. Thus, collectively, overwhelming evidence suggests versican to be a key regulator of NCC migration while it’s cleavage by ADAMTS-PGases permit a favourable environment for cell survival once NCCs have reached their destination and subsequently colonise.

Mesoderm

The inner mesodermal layer of the embryo is further divided into the lateral plate mesoderm, from which limb buds grow outwards from the body (proximal-distal), and bone anlage form from mesenchymal condensations within. Somatic and splanchnic mesoderm, the former associated with body wall structures and the appendicular skeleton, and the latter forming connective tissue surrounding gut-derived structures, are created in the lateral plate mesoderm during body folding and separated by the intraembryonic coelom. The intermediate mesoderm differentiates into the kidney and gonads, i.e. the urogenital system, whilst cranially located cardiogenic mesoderm forms the heart fields before body folding occurs, and further differentiates into the heart tubes that undergo looping and septation within the pericardial cavity.

Hyalectans and the Musculoskeletal System

Limbs Versican expression patterns were discovered in the developing limb by use of a LacZ surrogate marker for versican mRNA expression in the hdf (heart defect) heterozygous The Function of the Hyalectan Class of Proteoglycans … 61 mouse. The hdf mouse allele has a homozygous lethal chromosomal 13 insertion that was mapped to the Cspg2 (Vcan) gene. Homozygous hdf embryos die at E10.5 of a heart defect, described in detail forthwith in this chapter. In the developing mouse limb, versican (LacZ surrogate) mRNA is expressed in pre-cartilage condensations and nascent cartilage, and diminishes during cartilage maturation [53]. Versican expression is gradually replaced by aggrecan, as shown in the tibial cartilage diaphysis, although versican staining is still present at the epiphyseal end at E15 in the mouse. Hapln1 is differentially expressed, and whether versican aggregates along a HA scaffold in mesenchymal condensations is questionable due to notably reduced regional levels of HA [54]. Versican is also highly expressed in synovial joint interzones [55], where the ADAMTS-PGase cleavage site DPEAAE (Glu441↓Ala442 – V1 versican) is also readily detected in E12-16 day mouse embryos [56] suggesting that the remodelling of versican is necessary for synovial joint maturation. In the chicken, versican is also highly expressed in pre-cartilaginous mesenchyme and developing synovial joints and the knockdown of versican in presumptive ulna compromises mesenchymal condensation leading to reduced distal ulna length [57] while knock-down in the limb significantly reduces the area of joint interzones [58]. In a mouse model whereby versican deficiency is driven under the mesenchymal transcription factor Prx1 promoter, severely distorted digits are formed along with hypertrophic chondrocyte nodules in cartilage and a decrease in TGF-β incorporation in the joint interzone suggesting that versican may localise TGF-β, regulating its signalling during skeletal morphogenesis [59]. The growth plate is located at the epiphyses of endochondral (long) bones and elongates by columnar chondrocyte proliferation (the proliferative zone) during growth. Normally, chondrocytes within the growth plate secrete an aggrecan rich matrix that binds to HA via HAPLN1. CD44 in-turn retains HA on the cell surface creating an aggrecan and collagen II rich pericelluar matrix [60]. CD44 is up-regulated at later stages of limb development in the chicken, corresponding to the onset of chondrogenesis [61]. Heritable defects in the aggrecan gene (acan) lead to premature death and severe dwarfism in both chicken and mouse embryos [62]. In the nanomelic chicken mutant (nm), whereby acan is functionally disrupted by a premature stop codon in exon 12 [34], the growth plate is devoid of matrix. The absence of matrix is associated with altered bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Indian hedgehog (Ihh) signalling at the onset of growth plate differentiation [62]. In the case of limb skeletal muscle development large proteoglycans including versican can be found during early stages of myogenesis in the chicken limb, whilst in the later stages versican and small proteoglycans such as decorin are present in pericellular locations surrounding myotubes [63]. Adamts5 expression is closely associated with fusing myotubes in the E13.5 mouse embryo [49] suggesting versican proteolysis may be a fundamental process for embryonic myogenesis. In the chicken hindlimb, the addition of V0 and V1 versican leads to severe disturbances of nerve patterning through an apparent disruption of guidance cues. Low concentrations of V0 and V1 versican suppresses axon extension during nerve innervations, whilst at higher concentrations, causes growth cone collapse and rapid retraction along with untimely defasciculation and axon stalling both in vitro and in ovo [64]. Adamts5 is also expressed in pre-Schwann cells in developing mouse hindlimb nerves at E13.5 and at the site of neuromuscular junction formation [49]. During pentameric digit formation in the mouse limb, the generation of the G1-DPEAAE fragment of versican (V1) by ADAMTS-PGases is required to potentiate BMP-mediated apoptosis in interdigital mesenchyme [65]. 62 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Spine In the developing human spine, versican is expressed in intervertebral joint spaces whilst aggrecan and perlecan are co-localised in the rudiments of the vertebral body. V0 versican is the predominant species in the annular lamellae of annulus fibrosus and versican expression in the nucleus pulposus, a structure that is derived from the notochord, is devoid of its G3 domain, suggesting proteolytic removal or alternative splicing during de novo synthesis [66]. In the rat, aggrecan is expressed early in the nucleus pulposus, perhaps reflecting the preceding notochordal aggrecan expression described above in this chapter, and later in the inner annulus. Versican is also expressed later in the inner annulus and in the dorsal region of the outer annulus suggesting distinct roles for aggrecan and versican in the developing rat spine [67]. Aggrecan, neurocan and brevican are all found in the adult rat spinal cord [68]. In the Has2 conditional knockout mouse [under the control of the type II collagen (Col2a-1) promoter] both soft and hard tissue elements are affected; endochondral ossification is strikingly impaired, and remnants of the notochord are present in the spine. Consequently, Has2 conditional knockout mice die at E18.5 and present with impaired long bone formation and a lack of a growth plate, although aggrecan is still present [69-70].

Craniofacial Development Ihh signalling and associated aggrecan expression remains important throughout postnatal skeletal growth. For example, in a mouse model whereby Ihh is ablated postnatal, disruptions in temporomandibular joint (TMJ) growth and organisation are observed along with decreased Runx2, Sox9 Collagen I and II and aggrecan expression, all transcription factors and ECM molecules necessary for normal bone and joint growth and morphogenesis [71]. In rat mandibles (as well as the hind limbs), versican is abundant in woven bone (primary or immature bone tissue) but decreases in lamellar bone (secondary bone tissue) while its mRNA and cognate protein is also expressed by osteoblasts. Adamts1, -4 and -5 mRNA spatiotemporal pattern in the developing cranium is comparable to that of versican suggesting a requirement for dynamic remodelling by ADAMTS-PGases during craniofacial development [72]. The morphogen Shh can induce the expression of versican in cranial mesoderm and Shh gene expression regulates the positioning of the trigeminal ganglion [73], a structure that also expresses abundant Adamts5 mRNA in the mouse [49]. The developing TMJ predominately expresses the V1 isoform of versican in the rat and the core protein increases postnatally until P14. Versican is most abundant in the anterior and posterior peripheral attachments and less so in the central part of the discs of the TMJ [74]. Dermacan (vcanb), the zebrafish orthologue of V0 versican, mRNA is expressed in dermal bones, the sclerotome, tail fin bud, pharyngular arch primordial region, and otic vesicles as well as craniofacial bones including the opercle and dentary (dermal bones) in the developing zebrafish skull. Aggrecan expression, in contrast, is observed in the elements of craniofacial cartilage bones. Upon silencing of dermacan in the developing zebrafish, dermal bones, e.g. the opercle, dentary, and branchiostegal rays, as well as axial skeleton in the trunk show decreased ossification [75]. The pharyngeal arches of the zebrafish, which contribute to facial elements including the jaw, the telencephalon and the otic vesicles - the prospective ears, all express hapln1, which co-localises with both aggrecan and dermacan at 4 days post fertilisation (dpf) concurrent with chondrogenesis [38]. In the salmon, aggrecan is the predominant proteoglycan in nasal cartilage and its primary amino acid sequence is similar to the predicted pufferfish, zebrafish, bovine and human aggrecan sequence, all of which The Function of the Hyalectan Class of Proteoglycans … 63 possess the signature G1, G2 and G3 domains. The GAG attachment domains; KS and CS found in mammalian aggrecan are however absent in the salmon although serine-glycine or glycine-serine sequences are present [76]. Dynamic versican expression patterns during palatal shelf development has been noted in the mouse [77] and Adamts9+/-;bt/bt mice, which in addition to their pigmentation defect described previously in this chapter, present with a fully-penetrant secondary cleft palate, which is perinatal lethal in the mouse. Reduced versican processing in palatal shelf mesenchyme is a key feature of the Adamts9+/-;bt/bt mouse embryo at E13.5 / E14.5 and hdf haploinsufficiency on a bt/bt background, i.e. hdf+/-;bt/bt, which reduces the amount of versican expression by half, results in a greater penetrance of cleft palate than that observed on the bt/bt background alone [78] strongly suggesting that the ADAMTS-PGase generated G1-DPEAAE versican fragment is necessary for palatal shelf closure rather than the passive clearance of versican. In the developing mouse tooth germ versican mRNA expression is noted from E11.5 through E18.5 whilst expression in dental epithelium is first observed at E12, continuing through to the bell stage where NCC-derived odontoblasts, the cells that will eventually form and mineralise dentine, differentiation is induced. Immunohistochemical studies have shown versican expression in the stellate reticulum from bud to apposition stage as well as significant expression in the epithelium of developing teeth [79]. Proteoglycans other than versican that have been identified in the mouse tooth germ include decorin, biglycan, glypican, syndecan-1, and syndecan-3 although their patterns of expression vary throughout developmental stages E14.5 – E18.5 [80]. Partially erupted bovine incisors are also rich in versican, the major proteoglycan isolated after guanidine extraction, and HAPLN1 is also present and co-localised with versican to the dental pulp [81].

Hyalectans and Cardiac Development The dynamic regulation of versican in the developing mouse heart is noted whereby versican expression begins in a uniform manner in the heart tube but becomes predominantly localised to the right ventricular chamber prior to septation [82]. Notable expression is found in the endocardial cushions, structures which precede atrial and ventricular septation and the formation of the atrioventricular valves, and in the cardiac outflow tract. Fibulin-1 and fibulin-2 are also both prominent components of endocardial cushions [83]. Fibulin-1 is a versican binding partner that enhances the cleavage of versican by ADAMTS5 [65] whilst fibulin-2 binds all hyalectan C-type lectin domains except for neurocan [84]. In the chicken, contrary to the mouse, aggrecan is present in developing valve leaflets [85]. Venous, semilunar and atrioventricular valves also represent sites of significant versican expression in the mouse [82]. Versican is found on the atrial side whilst collagen fibres are found on the ventricular side of heart valves one-week after birth [86], reflecting the respective hydraulic pressure and elasticity requirements of circulating blood through the atria and ventricles, i.e. valvular back pressure is resisted by collagen II. The ADAMTS-PGase mediated G1-DPEAAE cleavage pattern is also asymmetrically distributed in the developing mouse heart, with the highest versicanase activity occurring subjacent to the endocardium [86]. Adenoviral over-expression of the V3 isoform of versican, which is devoid of GAG modifications (Figure 2), increases the thickness of proximal outflow tract myocardial layer in a proliferation-independent manner. In contrast, over- expression of versican G1 domain results in thinning and interruptions of the outflow tract 64 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. layer. V3 versican is able to promote myocardial cell-cell association while the versican G1 domain causes a loss of myocardial cell-cell association in the mouse [87] indicating differential roles for each of the globular domains present in versican during cardiac morphogenesis. The hdf mouse mentioned previously in this chapter provided the first definitive evidence of the functional consequence of versican deficiency during early mouse embryogenesis. The hdf knockout mouse dies by E10.5 and presents with A-P cardiac axis defects, absent endocardial cushions and the absence of the prospective right ventricle [88]. Crtl1 (Hapln1), versican, and HA are all co-expressed in the endocardial lining of the developing heart and also in mesenchyme of the atrioventricular junction and outflow tract, both of which are derived from the endocardium. At later stages of development, their co-expression is restricted to discrete populations of endocardially-derived mesenchyme. The Crtl1-deficient mouse also presents with several cardiac anomalies including atrioventricular septal and myocardial defects alongside a significant reduction in versican, not unlike that seen in the hdf mouse [89] indicating the requirement for a functionally intact versican-CRTL1/HAPLN1 network. In both the chicken and the mouse Sox9 is the predominant transcription factor regulating mesenchymal expansion and organisation of the ECM during valvulogenesis [90]. The absence of cleaved versican in the developing heart of the Adamts5 knockout mouse coincides with myxomatous heart valves, as well as increased SOX9 and BMP2 signalling in cardiac mesenchyme, and an increase in mesenchymal cell proliferation index. Versican haploinsufficiency on the Adamts5 knockout mouse background (Adamts5-/-;hdf+/-) partially rescues the myxomatous valve phenotype, again suggesting the clearance of versican is required for physiological cardiac morphogenesis [91]. Reduced versican cleavage is also observed in the Adamts9 haploinsufficient mouse heart (Adamts9+/-) where associated valvular and aortic anomalies and myxomatous valve degeneration occurs. In addition, chondrogenic nodules are found in the Adamts9+/- heart, suggesting a re-programming of chondrogenesis in the absence of cleaved versican [92]. In the Ets1 knockout mouse, NCCs fail to migrate to the proximal outflow tract of the heart, and prematurely differentiate into chondrogenic nodules rich in aggrecan [93] whilst dominant negative Ets1 expression inhibits chondrogenesis in the mouse [94]. It is interesting to speculate that ADAMTS9 might be required for complete NCC migration into the heart through versican-dependent proteolytic mechanisms, given the similarities in phenotypes between the Ets1 knockout mouse, the Adamts9 heterozygote mouse, and the prohibitive barrier to which versican confers upon NCC migration discussed earlier in this chapter. Studies in the mouse have established the homeobox transcription factor Tbx20 to be a potent regulator of heart development through the repression of Tbx2, which in-turn is thought to antagonise the transcriptional activity of Tbx5 [95]. Tbx20 adenoviral gain of function in chicken endocardial cells decreases aggrecan and versican expression and increases endocardial cell proliferation, whilst siRNA-mediated silencing of Tbx20 concordantly increases aggrecan and versican expression and decreases proliferation [96]. In the zebrafish, versican (vcana, versican G3 domain orthologue), bmp4, tbx5 and tbx20 are all expressed throughout heart development, and their expression is up-regulated when the tinman-related gene Nkx2.7 and Nkx2.5 is knocked down. Consequently, heart looping fails and the zebrafish two-chambered heart develops abnormally [97]. The N-myc downstream- regulated gene ndrg4 restricts versican (vcana) and bmp4 expression to the atrioventriclular The Function of the Hyalectan Class of Proteoglycans … 65 canal in zebrafish [98]. Interstingly, celecoxib (Celebrex) treated zebrafish embryos have reduced versican (vcana) and bmp4 during heart development, a condition also associated with the absence of heart valves and abnormal looping [99]. The zebrafish HAS2 orthologue (has2) is expressed in cardiac progenitor cells (CPCs) and is required for heart tube rotation. has2 expressing CPCs migrate towards an asymmetric BMP signalling gradient early in development in the lateral plate mesoderm, a process which drives left-right signalling and ultimately leads to the asymmetric development of the heart [100]. Through the use of a zebrafish model that lacks mature micro RNAs (miRNAs), i.e. dicer mutants, has2 was found to be a target for miR-23 and the up-regulation of has2 leads to cardiac cushion expansion in dicer mutant zebrafish [101]. In the frog, Xhas2 is expressed in the heart anlage where XHAPLN3 maintains an integral HA matrix. Xhapln3 mRNA expression overlaps with Xhas2 and Xvcan in early tailbud (stage 23) and knockdown of either Xhapln3 or Xhas2 causes a heart deficiency [102]. Xhas1 synthesises 40-200 kDa HA and Xhas2 synthesises HA with a molecular mass greater than 1 million Daltons [31], similar to that of other vertebrates described earlier in this chapter. Interestingly, neurocan is widely expressed in the quail and can be found at stage 11 in the embryonic heart and vasculature, and specifically, in the dorsal mesocardium, myocardium and prior heart-forming fields [103]. In the pig at the age of two-days post natal, atrioventricular valves demonstrate greater versican, elastin and HA turnover than during the 3rd trimester [104]. Versican mRNA expression is also dynamic in the mouse aorta from E18.5 to 12 weeks postnatal [105] collectively suggesting versican remodelling is important in postnatal cardiac maturation in higher vertebrates.

Hyalectans and Urogenital Development Chondroitin sulphate is the most abundant GAG in the developing rat kidney, although HS is thought to play a more profound role during ureteric budding, the mesonephric duct protrusion that forms most of the prospective kidney, ex vivo independent of its FGF binding properties [106]. In the frog, versican is expressed in the developing pronephros and pronephric ducts [27]. The ADAMTS-PGase Adamts9 is expressed in developing kidney mesenchmyal cells in the mouse [107] while Adamts1 expression is noted in epithelial cells [108], proximal and distal convoluted tubules and the developing loop of Henle [109]. The ADAMTS-PGase Adamts1 knockout mouse presents with kidney dysgenesis that becomes apparent in adulthood where interstitial fibrosis is found in the both the cortical and medullary regions [109]. A further cooperative role for the ADAMTS-PGases is shown whereby the combinatorial knockout mouse Adamts1-/-;Adamts4-/- dies by P3 and shows marked renal medulla thinning [110]. Collectively, these observations suggest that whilst CS (and thus versican) per se is less critical for kidney development, its targeted proteolysis by the ADAMTS-PGases is indeed necessary for kidney morphogenesis and normal adult kidney function.

Endoderm The foregut bifurcates to form the lower respiratory tract, whereby the lung buds intrude into the pleuropericardial cavity, which is soon partitioned into the pleural cavity that encases the developing lungs and the pericardial cavity that encases the heart. The foregut also forms the pharynx, oesophagus, duodenum, stomach, liver, and from dorsal and ventral pancreatic 66 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. budding the pancreas is formed. The midgut and hindgut are segmented into the bowel, which is defined by the superior mesenteric artery and the inferior mesenteric artery, respectively.

Hyalectans in Lung Development Glucocorticoid receptor (GR) knockout mice die postnatally due to respiratory failure and present with upregulated versican, and one of its binding partners, midkine at E18 and P1. Both midkine and versican mRNA dramatically decreases between E16.5 and E17.5 in wildtype mouse lungs, however in the GR knockout mouse this decrease is not apparent [111]. Versican mRNA expression also follows that of Shh mRNA in developing mouse lung epithelium [112]. Versican is also the predominant CSPG in the sheep fetal lung and decreases through late gestation in a similar manner to that of HA [113]. Versican is also detected in all phases of primordial germ cell migration [114], which are thought to arise from yolk-sac associated endodermal cells. In the frog, Xhas2 is expressed in the embryonic gut and in the hepatic cavity [31].

The Central Nervous System The forebrain, midbrain and hindbrain (prosencephalon, mesencephalon and rhombencephalon respectively in mammals) arise from mesencephalic flexure and the brain hemispheres and associated ventricles can now form. Neurogenesis originates from the proliferation of neurogenic progenitor cells, derived from neuroectoderm, which differentiate into post-mitotic neurons. A complex interplay of cell-signalling between all three germ layers directs the formation of the eye, whose major structures are the optic nerve and layers of the retina along with associated ganglion layers.

Hyalectans and the CNS

Aggrecan In the rat brain, quantitative assays have shown a steady increase in aggrecan expression from E14 through to 8 months postnatal age where aggrecan increases steadily until 5 months [115]. Aggrecan species were found to be similar in E14, P1, and adult spinal cords with specific aggrecan neoepitopes, suggesting that aggrecan is cleaved by both ADAMTS and MMP proteases throughout the development of the spinal cord [68]. In the chicken aggrecan is expressed in the brain at day 7 with increased expression at day 13 followed by marked decrease after day 16 and no expression post-hatching. It is proposed that the expression of aggrecan represents a migration arrest mechanism to facilitate the establishment of neuronal nuclei in the telencephalon [32]. In the developing eye, aggrecan is expressed in the retina but is absent in the optic nerve of the mouse at E18, until expression is observed at P7, at which time it is also expressed in the ganglion layers and diffusely distributed in the outer retina [116]. In the rat, however, aggrecan and versican expression has been observed in both the optic nerve and retina at E16 whilst at P9, aggrecan and versican expression are intense in the inner and outer plexiform layers and ganglion cell layers, with diffuse staining in the inner plexiform [117]. Hapln1 is expressed in the zebrafish neural floor plate and also in rhombomeres, which demarcate the developing brain [38].

The Function of the Hyalectan Class of Proteoglycans … 67

Brevican Brevican was first identified in the rat and the cat (Felis catus) where its expression is developmentally regulated and restricted to the brain and CNS [118]. The brevican knockout mouse is viable and fertile although it has altered neurocan expression. There are no obvious impairments of excitatory and inhibitory synaptic transmission and no deficits in learning or memory [119]. Brevican expression follows a similar pattern to that of aggrecan in the rat brain [115], as described above, and is the most abundant hyalectan in the CNS. The up- regulation of brevican is coincidental with glial cell proliferation and/or motility. Isoforms of brevican identified in the rat brain include the full-length isoform B/b150 and an underglycosylated isoform B/b130, which undergoes calcium-independent cell membrane localisation [120]. Brevican is highly expressed in ventricular zones throughout the neuraxis and is first detected at E15 in the spinal cord, where its expression becomes progressively more rostral, i.e. it follows caudal-rostral (and ventral-dorsal) gradients. Expression of brevican parallels the generation of glial cells after peak neurogenesis and coincides with gliogenesis in the rat [118]. Brevican has also been identified in the spinal cord of the adult rat [68]. In the mouse, brevican is found in PNNs suggestive of a role in CNS fibre tract development. In the developing CNS of the frog, brevican is expressed in the 5th and 6th rhombomeres in the tail-bud stage, in addition to the rostral spinal cord and periventricular region of the brain in the tadpole stage [37].

Neurocan Neurocan-deficient mice are viable and fertile with apparently normal brain anatomy, and morphology. Further, the PNNs comprising an ECM rich microenvironment, which are thought to facilitate neurogenesis also appear normal. Mild deficits in synaptic plasticity may however exist, whereby maintenance of late-phase hippocampal long-term potentiation is reduced [121]. Neurocan is a promoter of olfactory neuron outgrowth and is first expressed in primary olfactory neurons at E11.5 in the mouse [122]. Neurocan has been characterised to bind several molecules including the cell adhesion molecule L1 [123] as well as binding to various structural ECM components including, heparin, tenascin-C and tenascin-R, which also bind the G3 domain of other hyalectans. In addition, neurocan also binds the growth and mobility factors FGF-2, heparin binding growth associated molecule (HB-GAM), and amphoterin [124]. In the developing rat brain, neurocan expression increases until postnatal development whereby it co-ordinately declines [115]. Rat hippocampal neuron and primary astrocyte co-cultures promote intact synapse formation with the emergence of PNNs comprising HA, brevican and neurocan. Removal of GAG chains using chondroitinase ABC leads to decreased amplitude and reduced charge of miniature excitatory postsynaptic currents and glutamate insensitivity, suggesting that CS chains rather than the core proteoglycan mediates synaptic responses [125]. Neuroblasts in the developing retina of the rat express low levels of neurocan at E14 and E16. Neurocan expression is readily detected by immunostaining from P7 through P14, where it is mostly confined to the inner and outer plexiform layers, whilst adult retinas show concordantly reduced levels of neurocan. Two species of retina-derived neurocan are detected (220 and 150 kDa) by immunoblotting after chondroitinase ABC treatment, both of which are differentially regulated during retinal development [126].

68 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Versican In the rat brain, the expression of versican splice variants comprising the GAG-alpha domain (V0 and V2 – Figure 2) is low during embryogenesis but increases up to 10-fold from P10 through to P100. Conversely, versican comprising the GAG-beta domain (V0 and V1 – Figure 2) doubles in expression between E14 and birth then declines until 2 months postnatal where it reaches a steady state between 2 and 8 months, representing a 90% reduction in expression compared to the embryonic brain [115]. The EGF-like repeats of the versican G3 domain signal through the EGF receptor (EGFR) and the phosphorylation of Erk to promote neurite outgrowth in cultured embryonic rat hippocampal neurons, and also enhance glutamate receptor-mediated currents. Interestingly, the G3 domain is able to promote cell attachment independent of EGFR signalling [127]. The Pax6 haploinsufficient mouse has perturbed lens development and homeostasis while telencephalic development is impaired in the homozygous null Pax6 (Pax6-/-) mice, which is incompatible with lens development. Versican is also differentially regulated during lens development in the Pax6 deficient mouse suggesting that the versican gene (Cspg2/Vcan) promoter may have Pax6 binding elements [128]. The versican gene promoter is also under the signalling and transcriptional control of platelet-derived growth factor (PDGF) in smooth muscle cells, the PI3K-PKB pathway; Shh; Pax3; B-catenin/TCF; p53 and Wnt [129], some of which have roles in CNS development beyond the scope of this chapter. The rat cornea abundantly expresses CS at P1, which is undetectable by P14. Versican mRNA is initially highly expressed in the cornea but is barely detectable by P42 while HA is found in high levels early in development in the retina and declines thereafter in adulthood [130]. In the chicken, versican is expressed in the inner surface of neural retina at day 5 and by day 7 is strongly expressed in the nerve fibre and expression within the inner plexiform layer is apparent. Versican immunostaining is maximal at day 14 and then declines in the adult. Northern blot analysis shows the presence a 13 kb transcript representing the mRNA for translation of the 550 kDa core protein of the V0 versican isoform, whose bands are absent by day 20 [131]. HA is also expressed in the same area as versican in the developing retina of the chicken [132]. The V2 isoform of versican is a major inhibitor of neuronal growth of the mature CNS and is found in myelinated fibre tracts of the mouse brain and optic nerve. Its prohibitive growth properties are due to its core protein rather than its GAG attachments and it uses laminin-1 as a permissive substrate [133].

Pregnancy

Versican in Pregnancy

In the mouse uterus versican and HA are abundant in the endometrial stroma, however after implantation HA disappears whereas versican remains localised [134]. ADAMTS5 is also expressed at high levels in the human decidua and was first coined “implantin” where its mRNA was shown to be upregulated by interleukin-1β and down-regulated by TGF-β [135]. Mature oocytes express lower levels of versican in their cumulus, and pregnancy prediction relies on VCAN (and SDC4) expression in humans [136]. Versican cleavage is undetectable in Adamts1-/- mouse ovaries and the processing of versican is necessary for ovulation whereby The Function of the Hyalectan Class of Proteoglycans … 69 versican is normally cleared beyond the expected time of ovulation however it accumulates in Adamts1-/- mice [137], which are largely infertile. Upon in vitro maturation in the mouse, follicle stimulating hormone (FSH) and EGF both reduce levels of Vcan and Adamts1 during the maturation of mouse cumulus oocyte complex (COC) when compared to in vivo maturation, and Has2 is upregulated [138] indicating the necessity for versican remodelling and HA expression in the COC.

Dissecting Hyalectan Biology in the Zebrafish

The Zebrafish as a Model for Vertebrate Development

The zebrafish has recently emerged as an invaluable tool to dissect roles of mammalian gene orthologues throughout vertebrate embryonic development. Zebrafish embryos develop quickly with morphogenesis completed within 72 hours post fertilisation (hpf) [139]. Several hundred embryos can be acquired in a single lay, adding valuable statistical power to scientific studies. Perhaps the most profound property of the zebrafish embryo is its optical transparency, enabling the visualisation of internal structures using an inverted stereomicroscope. There are several advantages to using optically clear embryos whose development occurs ex utero; internal structures such as the looping heart tubes, the developing neural tube, notochord, somites and even myofibrils during somitogenesis, can be easily examined by whole-mount in situ hybridisation (WISH), or with good antibodies available, by whole-mount immunohistochemistry. The zebrafish embryo can be easily manipulated through the microinjection of either mRNA or morpholino antisense oligonucleotides (MOs) at the 1-8 cell stage after fertilisation, in order to study gene over- expression or knockdown (morphant embryos), respectively [140-141]. One advantage to the MO approach of gene silencing is the range of penetrance obtained; the resultant phenotype can be hypomorphic in nature or a complete knockdown within the same experiment revealing a variety of functions dependent upon gene-dosage. This advantage is particularly important when examining mammalian orthologues that are otherwise embryonic lethal early in development, i.e. Adamts9. However, the zebrafish can also be manipulated genetically, not unlike the mouse, to create stable gene knockout lines as well as transgenic fluorescent lines, which can also be engineered such that the fluorescent protein they express is under the control of tissue-specific gene promoters, a technique that truly exploits the optically transparent properties of the zebrafish embryo.

Expression and Function of Hyalectans in the Zebrafish

Earlier in this chapter examples of the expression and function of the zebrafish orthologues of versican (vcana and vcanb), HAS2 (has2), aggrecan (acan) and HAPLN1 (hapln1/crtl1) were given in the context of craniofacial development, cardiovascular development, and gastrulation/cell migration. In addition, the HAPLN genes have co-evolved with their respective proteoglycan partner, positioned back to back on the same chromosome suggesting that they arose from proteoglycan gene duplication in zebrafish [4] (and expanded 70 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. in a detailed analysis forthwith in this chapter). The purpose of the next section of this chapter is to highlight hyalectan biology by presenting new information regarding the evolution of the hyalectans, HAPLNs and ADAMTS-PGases across vertebrates and specifically in fish.

Figure 4. The evolution of the hyalectan/lectican family: aggrecan, brevican, neurocan and versican across vertebrates. The Function of the Hyalectan Class of Proteoglycans … 71

Figure 5. The evolution of the hyaluronan and proteoglycan link protein (HAPLN) family across vertebrates.

72 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 6. Synteny analysis illustrating the linkage of the hyalectan/lectican family: aggrecan, brevican, neurocan and versican, and hyaluronan and proteoglycan link proteins (HAPLNs) in the fish. Dr = Danio rerio, Ol = Oryzias latipes, Ga = Gasterosteus aculeatus, Tn = Tetraodon nigroviridis. Note that each HAPLN is linked in tandem to a respective hyalectan. 1R and 2R are the two whole genome duplications that occurred at the base of the Chordates, 3R is the teleost specific whole genome duplication.

The Evolutionary Conservation of the Hyalectans and Related Proteins

An extensive search for hyalectans and related gene sequences across vertebrates yielded exciting data regarding not only the evolution of the hyalectans (Figure 4) but also the co- evolution of their binding proteins, the HAPLNs [4] (Figure 5 and 6) across vertebrates. Homologous genes of the hyalectans were retrieved from version 64 of the Ensembl database (www.ensembl.org/). Retrieved homologous protein sequences were screened semi- automatically to remove redundancies and select representative species in groups of interest. Incomplete or poorly annotated sequences were discarded manually from protein alignments generated with Muscle v3.8.31 [142] (www.drive5.com/muscle). Total protein sequence alignments as well as large phylogenies are available upon request to Frédéric Brunet. Phylogenetic analyses were first generated using BIONJ [143], implemented in Seaview Version 4.2.12 [144] (pbil.univ-lyon1.fr/software/seaview.html). Maximum Likelihood phylogenetic trees were generated using PhyML (www.atgc-montpellier.fr/phyml/) [145] using JTT as substitution model and with a robustness calculated by 1000 bootstrap replicates. In addition, we used the software Genomicus v64.01 (www.dyogen.ens.fr/) to The Function of the Hyalectan Class of Proteoglycans … 73 localise the location of the duplicates onto the chromosomes and check their local synteny with neighbouring genes [146].

Aggrecan and Hapln3

Aggrecan ohnologous genes, that are paralogues originating from Whole Genome Duplication (WGD) (Figure 4), which represent acana and acanb respectively, are found in the Cypriniformes zebrafish Danio rerio (chromosomes 7 and 25) and the Percomorpha: medaka Oryzias latipes (chromosomes 3 and 6), stikleback Gasterosteus aculeatus (groups II and XIX) and tetraodon Tetraodon nigroviridis (chromosomes 5 and 13). Synteny analyses show that these genes originate from the Teleosts Specific Whole Genome Duplication (TSWGD or 3R) (Figure 6). Of note, acanb in fish is in head to head tandem position with hyaluronan and proteoglycan link protein 3 (hapln3 - ENSDARG00000045802), as it is in all tetrapod species. hapln3, is also duplicated however it is not found in the zebrafish, nor in the medaka (Figure 5). In the stickleback and the tetraodon, it is not associated with acana, but, very interestingly, a set of 4 genes in tandem are duplicated in the same strict order: rlbp1b, abhd2b, mfge8b and then hapln3, zebrafish (chromosome 25 only), medaka (chromosome 6 only), stickleback (groups XIX and II) and tetraodon (chromosomes 13 and 5), each representing hapln3a and hapln3b respectively. Note that even if hapln3b is not at the same location as acana, the genes are still on the same chromosomes (Figure 6).

Brevican and Hapln2

The brevican gene is also duplicated in fish, originating from TSWGD (Figure 4), which represent bcana and bcanb respectively. They are found in zebrafish (chromosome 16), medaka (chromosome 16), stickleback (scaffold 137 and group X) and tetraodon (unassigned and chromosome 21_random). bcana in fish is in head to head tandem position with hapln2 (ENSDARG00000003903) as it is in all tetrapod species (Figure 6). Even though there is no possibility to show the TSWGD at the chromosome level as no paralogue is found assigned for the two duplicates, both the phylogeny and the local synteny analysis (using Genomicus) are in favour of the TSWGD (Figure 5). The synteny analysis shows that acan and bcan are closely related with a total of 5 genes in common in the close neighbourhood of these genes (Figure 6).

Neurocan and Hapln4

The neurocan gene harbours paralogues that originated from the TSWGD (Figure 4): ncana and ncanb respectively, are found in zebrafish (chromosomes 2 and 22), medaka (chromosomes 17 and 4), stickleback (groups III and VIII) and tetraodon (chromosomes 15 and 1). ncanb in fish is in head to head tandem position with hapln4 (ENSDARG00000003903) (Figure 6) as is ncan and hapln4 in all tetrapod species. The genes cirbp, and gatad2 and an unknown gene are also duplicated nearby the ncan gene in the percomorphes, as in the zebrafish, it is sbno2, hmha1, polr2eb, gpx4a and rab11b that are 74 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. also duplicated. The phylogeny and the two synteny analyses (local and at the whole genome size) show that these genes come from the TSWGD (Figure 5).

Versican and Hapln1

Versican is also duplicated from the TSWGD (Figure 4): vcana and vcanb, respectively, are found in zebrafish (chromosome 5 and 10), medaka (chromosome 9 and 12) stickleback (groups XIII and XIV) and tetraodon (chromosomes 12 and 4). vcana is in head to head tandem position with hapln1a, and vcanb is in head to head tandem position with hapln1b (Figure 6) as it is in all tetrapod species for hapln1 and vcan. Two other genes are also duplicated in the neighbourhood of these two genes (there are mef2cb and arrdc3b). The phylogeny as well as the two synteny analyses shows that these genes originate from the TSWGD (Figure 5). Zebrafish versican (vcana) represents only a small portion of mammalian versican comprising its G3 globular domain. However, this domain is known to mediate important cellular events such as proliferation, and both resistance and sensitivity to apoptosis in tumourigenesis [147-152]. Dermacan (vcanb), on the other hand, shows homology to all regions of full-length human versican, i.e. V0, including the N-terminal region known to be proteolytically processed by the ADAMTS proteoglycanases. The specific ADAMTS– mediated versican cleavage site reported: DPEAAE↓A [16] is conserved in the zebrafish and is represented by the alternative cleavage site: VAEQE↓A. Whether ADAMTS-mediated remodelling of dermacan is apparent in the zebrafish throughout development is yet to be determined.

Rate of Evolution of the Hyalectans and Haplns

From these analyses it can be concluded that ncana evolves faster than ncanb and it is expected as ncanb is alongside hapln4, and ncana is not linked to a hapln gene anymore. In contrast, bcanb, which is not alongside any hapln does not appear to evolve faster than bcana. acanb evolves faster than acana and so to do the genes alongside them, i.e. hapln3b and hapln3a respectively. On the other hand, the evolutionary rates of the two other pairs are inverted, i.e. hapln1a, is evolving faster than its neighbouring gene vcana, and hapln1b is evolving slower than its neighbouring gene vcanb. Additionally, there is an acceleration of the evolutionary rate from eutherians for ncan and hapln4 and from metatherians for bcan and hapln2.

ADAMTS Proteoglycanases in the Zebrafish

The ADAMTS-PGase clade operates in cooperative proteolytic networks during embryogenesis, which is clearly apparent in combinatorial knockout mice presenting with phenotypes such as soft-tissue syndactyly and cleft palate with increased severity and/or penetrance observed when compared to individual knockouts [65, 78]. Due to the complexity of understanding cooperative ADAMTS biology in rodent models of development, we sought The Function of the Hyalectan Class of Proteoglycans … 75 to assess the suitability of zebrafish as a model to further elucidate ADAMTS-mediated mechanisms during embryogenesis. Further, previous comparative genomics studies have underscored the importance of ADAMTS enzymes throughout vertebrate evolution including the rapid expansion of this gene family concomitant with the emergence of chordates and vertebrates [153-155].

Figure 7. The evolution of the ADAMTS proteoglycanases across vertebrates. 76 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 8. (Continued). The Function of the Hyalectan Class of Proteoglycans … 77

Figure 8. (Continued). 78 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 8. Amino acid conservation of ADAMTS5 across vertebrates. The conserved furin cleavage site and catalytic motif are shown as gray boxes in the propeptide and peptidase domains respectively. Amino acid conservation legend: * = identical : = highly conserved . = conserved.

Figure 9. (Continued). The Function of the Hyalectan Class of Proteoglycans … 79

Figure 9. (Continued).

80 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 9. (Continued).

The Function of the Hyalectan Class of Proteoglycans … 81

Figure 9. (Continued).

82 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 9. Amino acid conservation of ADAMTS9 across vertebrates. The conserved furin cleavage site and catalytic motif are shown as gray boxes in the propeptide and peptidase domains respectively. Amino acid conservation legend: * = identical : = highly conserved . = conserved. Green amino acids = beginning of the disintegrin-like domain. The Function of the Hyalectan Class of Proteoglycans … 83

Figure 10. (Continued). 84 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

Figure 10. (Continued).

The Function of the Hyalectan Class of Proteoglycans … 85

Figure 10. Amino acid conservation of ADAMTS15 across vertebrates. The conserved furin cleavage site and catalytic motif are shown as gray boxes in the propeptide and peptidase domains respectively. Amino acid conservation legend: * = identical : = highly conserved . = conserved.

Further to the evolutionary analyses (Figure 7), we obtained bona fide full-length cDNA sequences for three ADAMTS proteoglycanase orthologues (adamts5 – Genbank accession: JF778846, adamts9 – Genbank accession: JF778848 and adamts15a – Genbank accession: JF778847). We next examined their conservation across vertebrate species and revealed a number of striking similarities; ADAMTS5, ADAMTS9 and ADAMTS15 primary amino acid sequences are highly homologous to their mammalian and avian orthologues, with the complete conservation of their zinc-binding catalytic motifs and their entire catalytic domain being highly conserved (Figure 8, 9 and 10 respectively) as well as the consensus furin cleavage site RXXR that facilitates the removal of the pro-domain (Figure 3) thus yielding an active metalloproteinase, as previously described for human ADAMTS5 [156], for example. Further, the eukaryotic post-translational modification consensus sequences for N- glycosylation, O-fucosylation and C-mannosylation (N, W and CSRTCDTG residues respectively) are also highly conserved in the zebrafish. However, zebrafish ADAMTS5 and ADAMTS9 and ADAMTS15 contain unique peptide sequences within their ancillary (C- terminal) domain. ADAMTS-PGases are likely conserved due to their importance during the establishment of the body plan and subsequent morphogenesis during vertebrate embryogenesis. Particularly evident in this study is the highly conserved functional modalities, including propeptide furin consensus sequence cleavage sites [156-159], N- glycosylation, O-fucosylation and C-mannosylation consensus sequences [160-161] and importantly, complete conservation of their catalytic motif across vertebrate (and invertebrate) species (Figure 11). The conservation of post-translational modifications suggests the function(s) of those modifications are conserved in the zebrafish. Two orthologues of FURIN: furina (sturgeon) and furinb have been characterised in the zebrafish [162], which interestingly have roles during craniofacial morphogenesis. The orthologue enzymes responsible for N-linked glycosylation (e.g. POMT1 and POMT2) are also present in the zebrafish genome [163] and when zpomt1 and zpomt2 are knocked down a range of 86 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al. phenotypes become apparent including an edematous pericardium and eye pigmentation defects as well as a twisted body [164]. There is also direct evidence of functional O- fucosylation [165] while the enzyme(s) responsible for C-mannosylation are yet to be identified in mammals [160]. The presence of a small unique peptide sequence disrupting the ancillary domains of both ADAMTS5 and ADAMTS15a suggests less selective pressure in this region of the genes.

Figure 11. Conservation of the zinc-dependent catalytic motif found in the ADAMTS proteoglycanases across species. Complete sequence conservation is seen for ADAMTS1, ADAMTS5 (except for Gallus gallus), and ADAMTS9 and ADAMTS15 suggesting substrate targeted homology throughout invertebrate and vertebrate evolution. Bolded numbers = amino acid position in each respective ADAMTS. Asterisks = predicted proteoglycanase sequences. The Function of the Hyalectan Class of Proteoglycans … 87

Conclusion

The hyalectans and their binding proteins, the HAPLNs, are well conserved in fish with constant evolution through WGD readily apparent. The duplication of these genes could lead to partial redundancy, or alternatively, a gain of function in fish that is not apparent in other vertebrates. There are several studies that have already utilised the zebrafish as a sound platform for dissecting the roles of the hyalectans and HAPLNs (and HA) during vertebrate embryogenesis, as outlined throughout this chapter. Equally, the ADAMTS-PGases show high conservation in the fish, and although adamts4 has been lost, adamts8 and adamts15 have undergone WGD (Figure 7), indicative of compensation for the loss of adamts4, but these gene duplications may also represent alternative functions. Further, remarkable conservation at the primary amino acid level across vertebrate species is shown for three of the ADAMTS-PGase family members whilst the same level of conservation exists for other predicted zebrafish adamts proteoglycanase sequences, i.e. ADAMTS1 and ADAMTS8a. Given the incontrovertible roles of the ADAMTS proteoglycanase genes during mammalian (vertebrate) embryonic development described throughout this chapter, and the profound roles demonstrated by their substrate versican, it is likely that similar and additional roles exist in the developing zebrafish embryo. The developing zebrafish comprises highly conserved vertebrate embryonic processes and a functional network consisting of the hyaluronic acid synthases (and therefore HA), hyalectans, HAPLNs and ADAMTS-PGases. Thus, given the presence and conservation of these genes, presented in the final section of this chapter, and its advantages as a developmental model, the zebrafish represents an attractive alternative to mammals for elucidating yet undiscovered roles of the “hyalectan/lectican-developmental cooperative network” (H/L-DCN).

Author Contributions

FB performed the evolutionary and phylogenetic analyses and constructed the evolutionary trees and synteny analysis figures, as well as writing commentary regarding the subsequent findings and critiquing the manuscript. CK performed experiments confirming adamts proteoglycanase mRNA expression in zebrafish embryos, edited the manuscript and assisted with literature searches. ADS performed the majority of the experiments confirming full-length adamts proteoglycanase mRNA expression in zebrafish embryos and constructed the amino-acid alignment figures as well as the mapping of the post-translational modifications and other features of the ADAMTS proteoglycanase clade. DRM conceived the study, performed the literature review, constructed the remaining figures and wrote the manuscript.

References

[1] Funderburgh, J.L., Keratan sulfate: structure, biosynthesis, and function. Glycobiology, 2000. 10(10): p. 951-8. 88 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

[2] Iozzo, R.V. and A.D. Murdoch, Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J, 1996. 10(5): p. 598-614. [3] Lee, J.Y. and A.P. Spicer, Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curr Opin Cell Biol, 2000. 12(5): p. 581-6. [4] Spicer, A.P., A. Joo, and R.A. Bowling, Jr., A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes: the missing links. J Biol Chem, 2003. 278(23): p. 21083-91. [5] Fosang, A.J. and T.E. Hardingham, Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein. Biochem J, 1989. 261(3): p. 801-9. [6] Day, J.M., et al., Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C and other extracellular matrix proteins. J Biol Chem, 2004. 279(13): p. 12511-8. [7] Nakada, M., et al., Human glioblastomas overexpress ADAMTS-5 that degrades brevican. Acta Neuropathol, 2005. 110(3): p. 239-46. [8] Naso, M.F., et al., Expression pattern and mapping of the murine versican gene (Cspg2) to chromosome 13. Genomics, 1995. 29(1): p. 297-300. [9] Apte, S.S., A disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms. J Biol Chem, 2009. 284(46): p. 31493-7. [10] Gendron, C., et al., Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4. J Biol Chem, 2007. 282(25): p. 18294-306. [11] Kintakas, C. and D.R. McCulloch, Emerging roles for ADAMTS5 during development and disease. Matrix Biol, 2011. 30(5-6): p. 311-7. [12] Zeng, W., et al., Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: comparative analyses with ADAMTS-5, -9, -16 and -18. Biochim Biophys Acta, 2006. 1760(3): p. 517-24. [13] Glasson, S.S., et al., Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature, 2005. 434(7033): p. 644-8. [14] Stanton, H., et al., ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature, 2005. 434(7033): p. 648-52. [15] Kuno, K., et al., ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan. FEBS Lett, 2000. 478(3): p. 241-5. [16] Sandy, J.D., et al., Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4. J Biol Chem, 2001. 276(16): p. 13372-8. [17] Cross, A.K., et al., Differential expression of ADAMTS-1, -4, -5 and TIMP-3 in rat spinal cord at different stages of acute experimental autoimmune encephalomyelitis. J Autoimmun, 2006. 26(1): p. 16-23. [18] Chan, C.K., et al., Differentiation of cardiomyocytes from human embryonic stem cells is accompanied by changes in the extracellular matrix production of versican and hyaluronan. J Cell Biochem, 2010. 111(3): p. 585-96. The Function of the Hyalectan Class of Proteoglycans … 89

[19] Shukla, S., et al., Synthesis and organization of hyaluronan and versican by embryonic stem cells undergoing embryoid body differentiation. J Histochem Cytochem, 2010. 58(4): p. 345-58. [20] Koos, D.S. and R.K. Ho, The nieuwkoid gene characterizes and mediates a Nieuwkoop- center-like activity in the zebrafish. Curr Biol, 1998. 8(22): p. 1199-206. [21] Bischof, J. and W. Driever, Regulation of hhex expression in the yolk syncytial layer, the potential Nieuwkoop center homolog in zebrafish. Dev Biol, 2004. 276(2): p. 552- 62. [22] Sulek, K., [Nobel prize in 1935 for the discovery of the role of the organizers in the development of the organism]. Wiad Lek, 1968. 21(8): p. 708. [23] Smith, J.C. and J.M. Slack, Dorsalization and neural induction: properties of the organizer in Xenopus laevis. J Embryol Exp Morphol, 1983. 78: p. 299-317. [24] Keller, R., J. Shih, and A. Sater, The cellular basis of the convergence and extension of the Xenopus neural plate. Dev Dyn, 1992. 193(3): p. 199-217. [25] Keller, R., et al., Planar induction of convergence and extension of the neural plate by the organizer of Xenopus. Dev Dyn, 1992. 193(3): p. 218-34. [26] Little, C.B., et al., Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair. J Clin Invest, 2007. 117(6): p. 1627-36. [27] Casini, P., et al., Identification and gene expression of versican during early development of Xenopus. Int J Dev Biol, 2008. 52(7): p. 993-8. [28] Camenisch, T.D., et al., Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest, 2000. 106(3): p. 349-60. [29] Bakkers, J., et al., Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation. Development, 2004. 131(3): p. 525-37. [30] Toole, B.P., Hyaluronan in morphogenesis. J Intern Med, 1997. 242(1): p. 35-40. [31] Koprunner, M., J. Mullegger, and G. Lepperdinger, Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhas1 and 2 during early development of Xenopus laevis. Mech Dev, 2000. 90(2): p. 275-8. [32] Schwartz, N.B., et al., Brain aggrecan. Perspect Dev Neurobiol, 1996. 3(4): p. 291-306. [33] Masuda, T., et al., Developmental regulation of notochord-derived repulsion for dorsal root ganglion axons. Mol Cell Neurosci, 2004. 25(2): p. 217-27. [34] Domowicz, M., et al., The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene. Dev Biol, 1995. 171(2): p. 655-64. [35] Yamagata, M., T. Shinomura, and K. Kimata, Tissue variation of two large chondroitin sulfate proteoglycans (PG-M/versican and PG-H/aggrecan) in chick embryos. Anat Embryol (Berl), 1993. 187(5): p. 433-44. [36] Smits, P. and V. Lefebvre, Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs. Development, 2003. 130(6): p. 1135-48. [37] Sander, V., J. Mullegger, and G. Lepperdinger, Xenopus brevican is expressed in the notochord and the brain during early embryogenesis. Mech Dev, 2001. 102(1-2): p. 251-3. 90 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

[38] Kang, J.S., et al., Molecular cloning and developmental expression of a hyaluronan and proteoglycan link protein gene, crtl1/hapln1, in zebrafish. Zoolog Sci, 2008. 25(9): p. 912-8. [39] Colas, J.F. and G.C. Schoenwolf, Localization of cartilage linking protein 1 during primary neurulation in the chick embryo. Brain Res Dev Brain Res, 2003. 141(1-2): p. 141-8. [40] Sorrell, J.M., et al., Versican in human fetal skin development. Anat Embryol (Berl), 1999. 199(1): p. 45-56. [41] Soma, T., M. Tajima, and J. Kishimoto, Hair cycle-specific expression of versican in human hair follicles. J Dermatol Sci, 2005. 39(3): p. 147-54. [42] Feng, M., G. Yang, and J. Wu, Versican targeting by RNA interference suppresses aggregative growth of dermal papilla cells. Clin Exp Dermatol, 2011. 36(1): p. 77-84. [43] Dutt, S., et al., Versican V0 and V1 guide migratory neural crest cells. J Biol Chem, 2006. 281(17): p. 12123-31. [44] Le Douarin, N.M., C. Ziller, and G.F. Couly, Patterning of neural crest derivatives in the avian embryo: in vivo and in vitro studies. Dev Biol, 1993. 159(1): p. 24-49. [45] Conway, S.J., et al., Neural crest is involved in development of abnormal myocardial function. J Mol Cell Cardiol, 1997. 29(10): p. 2675-85. [46] Poelmann, R.E. and A.C. Gittenberger-de Groot, A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development? Dev Biol, 1999. 207(2): p. 271-86. [47] St Amand, T.R., J.T. Lu, and K.R. Chien, Defects in cardiac conduction system lineages and malignant arrhythmias: developmental pathways and disease. Novartis Found Symp, 2003. 250: p. 260-70; discussion 271-5, 276-9. [48] Dutt, S., et al., Guidance of neural crest cell migration: the inhibitory function of the chondroitin sulfate proteoglycan, versican. ScientificWorld Journal, 2006. 6: p. 1114-7. [49] McCulloch, D.R., et al., Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues. Gene Expr Patterns, 2009. 9(5): p. 314-23. [50] Silver, D.L., et al., The secreted metalloprotease ADAMTS20 is required for melanoblast survival. PLoS Genet, 2008. 4(2): p. e1000003. [51] Perissinotto, D., et al., Avian neural crest cell migration is diversely regulated by the two major hyaluronan-binding proteoglycans PG-M/versican and aggrecan. Development, 2000. 127(13): p. 2823-42. [52] Henderson, D.J., P. Ybot-Gonzalez, and A.J. Copp, Over-expression of the chondroitin sulphate proteoglycan versican is associated with defective neural crest migration in the Pax3 mutant mouse (splotch). Mech Dev, 1997. 69(1-2): p. 39-51. [53] Yamamura, H., et al., A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse. Dev Biol, 1997. 186(1): p. 58-72. [54] Shibata, S., et al., In situ hybridization and immunohistochemistry of versican, aggrecan and link protein, and histochemistry of hyaluronan in the developing mouse limb bud cartilage. J Anat, 2003. 203(4): p. 425-32. [55] Shepard, J.B., et al., Versican expression during synovial joint morphogenesis. Int J Biol Sci, 2007. 3(6): p. 380-4. [56] Capehart, A.A., Proteolytic cleavage of versican during limb joint development. Anat Rec (Hoboken), 2010. 293(2): p. 208-14. The Function of the Hyalectan Class of Proteoglycans … 91

[57] Shepard, J.B., et al., Versican knock-down compromises chondrogenesis in the embryonic chick limb. Anat Rec (Hoboken), 2008. 291(1): p. 19-27. [58] Nagchowdhuri, P.S., et al., Versican Knockdown Reduces Interzone Area During Early Stages of Chick Synovial Joint Development. Anat Rec (Hoboken), 2011. [59] Choocheep, K., et al., Versican facilitates chondrocyte differentiation and regulates joint morphogenesis. J Biol Chem, 2010. 285(27): p. 21114-25. [60] Heinegard, D. and T. Saxne, The role of the cartilage matrix in osteoarthritis. Nat Rev Rheumatol, 2011. 7(1): p. 50-6. [61] Rousche, K.T. and C.B. Knudson, Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid. Matrix Biol, 2002. 21(1): p. 53-62. [62] Domowicz, M.S., et al., Aggrecan modulation of growth plate morphogenesis. Dev Biol, 2009. 329(2): p. 242-57. [63] Carrino, D.A., J.M. Sorrell, and A.I. Caplan, Dynamic expression of proteoglycans during chicken skeletal muscle development and maturation. Poult Sci, 1999. 78(5): p. 769-77. [64] Dutt, S., et al., Versican V0 and V1 direct the growth of peripheral axons in the developing chick hindlimb. J Neurosci, 2011. 31(14): p. 5262-70. [65] McCulloch, D.R., et al., ADAMTS metalloproteases generate active versican fragments that regulate interdigital web regression. Dev Cell, 2009. 17(5): p. 687-98. [66] Smith, S.M., et al., Topographical variation in the distributions of versican, aggrecan and perlecan in the foetal human spine reflects their diverse functional roles in spinal development. Histochem Cell Biol, 2009. 132(5): p. 491-503. [67] Hayes, A.J., M. Benjamin, and J.R. Ralphs, Extracellular matrix in development of the intervertebral disc. Matrix Biol, 2001. 20(2): p. 107-21. [68] Lemons, M.L., et al., Intact aggrecan and fragments generated by both aggrecanse and metalloproteinase-like activities are present in the developing and adult rat spinal cord and their relative abundance is altered by injury. J Neurosci, 2001. 21(13): p. 4772-81. [69] Moffatt, P., et al., Hyaluronan production by means of Has2 gene expression in chondrocytes is essential for long bone development. Dev Dyn, 2011. 240(2): p. 404-12. [70] Roughley, P.J., et al., The role of hyaluronan produced by Has2 gene expression in development of the spine. Spine (Phila Pa 1976), 2011. 36(14): p. E914-20. [71] Ochiai, T., et al., Indian hedgehog roles in post-natal TMJ development and organization. J Dent Res, 2010. 89(4): p. 349-54. [72] Nakamura, M., et al., Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb. J Histochem Cytochem, 2005. 53(12): p. 1553-62. [73] Fedtsova, N., R. Perris, and E.E. Turner, Sonic hedgehog regulates the position of the trigeminal ganglia. Dev Biol, 2003. 261(2): p. 456-69. [74] Toriya, N., et al., Expression and localization of versican during postnatal development of rat temporomandibular joint disc. Histochem Cell Biol, 2006. 125(3): p. 205-14. [75] Kang, J.S., et al., Characterization of dermacan, a novel zebrafish lectican gene, expressed in dermal bones. Mech Dev, 2004. 121(3): p. 301-12. [76] Kakizaki, I., et al., Identification of proteoglycan from salmon nasal cartilage. Arch Biochem Biophys, 2011. 506(1): p. 58-65. 92 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

[77] Vaziri Sani, F., et al., Developmental changes in cellular and extracellular structural macromolecules in the secondary palate and in the nasal cavity of the mouse. Eur J Oral Sci, 2010. 118(3): p. 221-36. [78] Enomoto, H., et al., Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation. Development, 2010. 137(23): p. 4029-38. [79] Jiang, B.Z., et al., Expression, localisation and synthesis of versican by the enamel organ of developing mouse molar tooth germ: an in vivo and in vitro study. Arch Oral Biol, 2010. 55(12): p. 995-1006. [80] Hikake, T., et al., Comparison of expression patterns between CREB family transcription factor OASIS and proteoglycan core protein genes during murine tooth development. Anat Embryol (Berl), 2003. 206(5): p. 373-80. [81] Yamauchi, S., et al., Identification, partial characterization, and distribution of versican and link protein in bovine dental pulp. J Dent Res, 1997. 76(11): p. 1730-6. [82] Henderson, D.J. and A.J. Copp, Versican expression is associated with chamber specification, septation, and valvulogenesis in the developing mouse heart. Circ Res, 1998. 83(5): p. 523-32. [83] Miosge, N., et al., Ultrastructural localization of microfibrillar fibulin-1 and fibulin-2 during heart development indicates a switch in molecular associations. Cell Mol Life Sci, 1998. 54(6): p. 606-13. [84] Olin, A.I., et al., The proteoglycans aggrecan and Versican form networks with fibulin- 2 through their lectin domain binding. J Biol Chem, 2001. 276(2): p. 1253-61. [85] Lincoln, J., C.M. Alfieri, and K.E. Yutzey, BMP and FGF regulatory pathways control cell lineage diversification of heart valve precursor cells. Dev Biol, 2006. 292(2): p. 292-302. [86] Kruithof, B.P., S.A. Krawitz, and V. Gaussin, Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling. Dev Biol, 2007. 302(1): p. 208-17. [87] Kern, C.B., et al., Versican proteolysis mediates myocardial regression during outflow tract development. Dev Dyn, 2007. 236(3): p. 671-83. [88] Mjaatvedt, C.H., et al., The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev Biol, 1998. 202(1): p. 56-66. [89] Wirrig, E.E., et al., Cartilage link protein 1 (Crtl1), an extracellular matrix component playing an important role in heart development. Dev Biol, 2007. 310(2): p. 291-303. [90] Lincoln, J., et al., Sox9 is required for precursor cell expansion and extracellular matrix organization during mouse heart valve development. Dev Biol, 2007. 305(1): p. 120-32. [91] Dupuis, L.E., et al., Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease. Dev Biol, 2011. 357(1): p. 152-64. [92] Kern, C.B., et al., Reduced versican cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic anomalies. Matrix Biol, 2010. 29(4): p. 304-16. [93] Gao, Z., et al., Ets1 is required for proper migration and differentiation of the cardiac neural crest. Development, 2010. 137(9): p. 1543-51. [94] Sugiura, K. and K. Ito, Roles of Ets-1 and p70S6 kinase in chondrogenic and gliogenic specification of mouse mesencephalic neural crest cells. Mech Dev, 2010. 127(3-4): p. 169-82. The Function of the Hyalectan Class of Proteoglycans … 93

[95] Stennard, F.A., et al., Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development, 2005. 132(10): p. 2451-62. [96] Shelton, E.L. and K.E. Yutzey, Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression. Dev Biol, 2007. 302(2): p. 376- 88. [97] Tu, C.T., T.C. Yang, and H.J. Tsai, Nkx2.7 and Nkx2.5 function redundantly and are required for cardiac morphogenesis of zebrafish embryos. PLoS One, 2009. 4(1): p. e4249. [98] Qu, X., et al., Ndrg4 is required for normal myocyte proliferation during early cardiac development in zebrafish. Dev Biol, 2008. 317(2): p. 486-96. [99] Xu, D.J., et al., Celecoxib impairs heart development via inhibiting cyclooxygenase-2 activity in zebrafish embryos. Anesthesiology, 2011. 114(2): p. 391-400. [100] Smith, K.A., et al., Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells. Dev Cell, 2008. 14(2): p. 287- 97. [101] Lagendijk, A.K., et al., MicroRNA-23 restricts cardiac valve formation by inhibiting Has2 and extracellular hyaluronic acid production. Circ Res, 2011. 109(6): p. 649-57. [102] Ito, Y., et al., XHAPLN3 plays a key role in cardiogenesis by maintaining the hyaluronan matrix around heart anlage. Dev Biol, 2008. 319(1): p. 34-45. [103] Mishima, N. and S. Hoffman, Neurocan in the embryonic avian heart and vasculature. Anat Rec A Discov Mol Cell Evol Biol, 2003. 272(2): p. 556-62. [104] Stephens, E.H., et al., Perinatal changes in mitral and aortic valve structure and composition. Pediatr Dev Pathol, 2010. 13(6): p. 447-58. [105] Adhikari, N., et al., Changes in expression of proteoglycan core proteins and heparan sulfate enzymes in the developing and adult murine aorta. J Cardiovasc Transl Res, 2011. 4(3): p. 313-20. [106] Steer, D.L., et al., Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney. Dev Biol, 2004. 272(2): p. 310-27. [107] Jungers, K.A., et al., Adamts9 is widely expressed during mouse embryo development. Gene Expr Patterns, 2005. 5(5): p. 609-17. [108] Thai, S.N. and M.L. Iruela-Arispe, Expression of ADAMTS1 during murine development. Mech Dev, 2002. 115(1-2): p. 181-5. [109] Mittaz, L., et al., Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis. Nephrol Dial Transplant, 2005. 20(2): p. 419-23. [110] Boerboom, D., et al., Partially redundant functions of Adamts1 and Adamts4 in the perinatal development of the renal medulla. Dev Dyn, 2011. 240(7): p. 1806-14. [111] Kaplan, F., et al., The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development. Am J Respir Cell Mol Biol, 2003. 28(1): p. 33-41. [112] Rutter, M., et al., Gli2 influences proliferation in the developing lung through regulation of cyclin expression. Am J Respir Cell Mol Biol, 2010. 42(5): p. 615-25. [113] Faggian, J., et al., Changes in versican and chondroitin sulfate proteoglycans during structural development of the lung. Am J Physiol Regul Integr Comp Physiol, 2007. 293(2): p. R784-92. 94 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

[114] Soto-Suazo, M., et al., Differential expression of glycosaminoglycans and proteoglycans in the migratory pathway of the primordial germ cells of the mouse. Histochem Cell Biol, 2002. 118(1): p. 69-78. [115] Milev, P., et al., Differential regulation of expression of hyaluronan-binding proteoglycans in developing brain: aggrecan, versican, neurocan, and brevican. Biochem Biophys Res Commun, 1998. 247(2): p. 207-12. [116] Ali, S.A., Y.Z. Hosaka, and M. Uehara, Spatiotemporal distribution of chondroitin sulfate proteoglycans in the developing mouse retina and optic nerve. J Vet Med Sci, 2011. 73(1): p. 13-8. [117] Popp, S., et al., Developmental changes of aggrecan, versican and neurocan in the retina and optic nerve. Exp Eye Res, 2004. 79(3): p. 351-6. [118] Jaworski, D.M., G.M. Kelly, and S. Hockfield, BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain. J Cell Biol, 1994. 125(2): p. 495-509. [119] Brakebusch, C., et al., Brevican-deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory. Mol Cell Biol, 2002. 22(21): p. 7417-27. [120] Viapiano, M.S., R.T. Matthews, and S. Hockfield, A novel membrane-associated glycovariant of BEHAB/brevican is up-regulated during rat brain development and in a rat model of invasive glioma. J Biol Chem, 2003. 278(35): p. 33239-47. [121] Zhou, X.H., et al., Neurocan is dispensable for brain development. Mol Cell Biol, 2001. 21(17): p. 5970-8. [122] Clarris, H.J., U. Rauch, and B. Key, Dynamic spatiotemporal expression patterns of neurocan and phosphacan indicate diverse roles in the developing and adult mouse olfactory system. J Comp Neurol, 2000. 423(1): p. 99-111. [123] Itoh, K., et al., Brain development in mice lacking L1-L1 homophilic adhesion. J Cell Biol, 2004. 165(1): p. 145-54. [124] Rauch, U., K. Feng, and X.H. Zhou, Neurocan: a brain chondroitin sulfate proteoglycan. Cell Mol Life Sci, 2001. 58(12-13): p. 1842-56. [125] Pyka, M., et al., Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons. Eur J Neurosci, 2011. 33(12): p. 2187-202. [126] Inatani, M., et al., Identification of a nervous tissue-specific chondroitin sulfate proteoglycan, neurocan, in developing rat retina. Invest Ophthalmol Vis Sci, 1999. 40(10): p. 2350-9. [127] Xiang, Y.Y., et al., Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor. J Biol Chem, 2006. 281(28): p. 19358-68. [128] Wolf, L.V., et al., Identification of pax6-dependent gene regulatory networks in the mouse lens. PLoS One, 2009. 4(1): p. e4159. [129] Rahmani, M., et al., Versican: signaling to transcriptional control pathways. Can J Physiol Pharmacol, 2006. 84(1): p. 77-92. [130] Koga, T., et al., Expression of a chondroitin sulfate proteoglycan, versican (PG-M), during development of rat cornea. Curr Eye Res, 2005. 30(6): p. 455-63. [131] Zako, M., et al., Transient expression of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retina. J Neurochem, 1997. 69(5): p. 2155-61. The Function of the Hyalectan Class of Proteoglycans … 95

[132] Inoue, Y., et al., Hyaluronan dynamics during retinal development. Brain Res, 2009. 1256: p. 55-60. [133] Schmalfeldt, M., et al., Brain derived versican V2 is a potent inhibitor of axonal growth. J Cell Sci, 2000. 113 ( Pt 5): p. 807-16. [134] San Martin, S., M. Soto-Suazo, and T.M. Zorn, Distribution of versican and hyaluronan in the mouse uterus during decidualization. Braz J Med Biol Res, 2003. 36(8): p. 1067- 71. [135] Zhu, H., P.C. Leung, and C.D. MacCalman, Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines. Hum Reprod, 2007. 22(1): p. 63-74. [136] Wathlet, S., et al., Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients. Hum Reprod, 2011. 26(5): p. 1035-51. [137] Brown, H.M., et al., ADAMTS1 cleavage of versican mediates essential structural remodeling of the ovarian follicle and cumulus-oocyte matrix during ovulation in mice. Biol Reprod, 2010. 83(4): p. 549-57. [138] Dunning, K.R., et al., Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod, 2007. 22(11): p. 2842-50. [139] Kimmel, C.B., et al., Stages of embryonic development of the zebrafish. Dev Dyn, 1995. 203(3): p. 253-310. [140] Lieschke, G.J. and P.D. Currie, Animal models of human disease: zebrafish swim into view. Nat Rev Genet, 2007. 8(5): p. 353-67. [141] Nasevicius, A. and S.C. Ekker, Effective targeted gene 'knockdown' in zebrafish. Nat Genet, 2000. 26(2): p. 216-20. [142] Edgar, R.C., MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 2004. 32(5): p. 1792-7. [143] Gascuel, O., BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol, 1997. 14(7): p. 685-95. [144] Gouy, M., S. Guindon, and O. Gascuel, SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol, 2010. 27(2): p. 221-4. [145] Guindon, S., et al., New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol, 2010. 59(3): p. 307- 21. [146] Muffato, M., et al., Genomicus: a database and a browser to study gene synteny in modern and ancestral genomes. Bioinformatics, 2010. 26(8): p. 1119-21. [147] LaPierre, D.P., et al., The ability of versican to simultaneously cause apoptotic resistance and sensitivity. Cancer Res, 2007. 67(10): p. 4742-50. [148] Miquel-Serra, L., et al., V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis. Lab Invest, 2006. 86(9): p. 889-901. [149] Wu, Y., et al., Versican protects cells from oxidative stress-induced apoptosis. Matrix Biol, 2005. 24(1): p. 3-13. [150] Sheng, W., et al., The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis. Mol Biol Cell, 2005. 16(3): p. 1330-40. [151] Wu, Y., et al., Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth. Mol Biol Cell, 2004. 15(5): p. 2093-104. 96 Frédéric Brunet, Christopher Kintakas, Adam D. Smith et al.

[152] Cattaruzza, S., et al., The globular domains of PG-M/versican modulate the proliferation-apoptosis equilibrium and invasive capabilities of tumor cells. FASEB J, 2004. 18(6): p. 779-81. [153] Huxley-Jones, J., et al., The characterisation of six ADAMTS proteases in the basal chordate Ciona intestinalis provides new insights into the vertebrate ADAMTS family. Int J Biochem Cell Biol, 2005. 37(9): p. 1838-45. [154] Huxley-Jones, J., et al., The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio. BMC Evol Biol, 2007. 7: p. 63. [155] Nicholson, A.C., et al., Functional evolution of ADAMTS genes: evidence from analyses of phylogeny and gene organization. BMC Evol Biol, 2005. 5: p. 11. [156] Longpre, J.M., et al., Characterization of proADAMTS5 processing by proprotein convertases. Int J Biochem Cell Biol, 2009. 41(5): p. 1116-26. [157] Koo, B.H. and S.S. Apte, Cell-surface processing of the metalloprotease pro- ADAMTS9 is influenced by the chaperone GRP94/gp96. J Biol Chem, 2010. 285(1): p. 197-205. [158] Longpre, J.M. and R. Leduc, Identification of prodomain determinants involved in ADAMTS-1 biosynthesis. J Biol Chem, 2004. 279(32): p. 33237-45. [159] Tortorella, M.D., et al., ADAMTS-4 (aggrecanase-1): N-terminal activation mechanisms. Arch Biochem Biophys, 2005. 444(1): p. 34-44. [160] Wang, L.W., et al., O-fucosylation of thrombospondin type 1 repeats in ADAMTS-like- 1/punctin-1 regulates secretion: implications for the ADAMTS superfamily. J Biol Chem, 2007. 282(23): p. 17024-31. [161] Wang, L.W., et al., Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan. J Biol Chem, 2009. 284(44): p. 30004-15. [162] Walker, M.B., et al., Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning. Dev Biol, 2006. 295(1): p. 194-205. [163] Moore, C.J., H.T. Goh, and J.E. Hewitt, Genes required for functional glycosylation of dystroglycan are conserved in zebrafish. Genomics, 2008. 92(3): p. 159-67. [164] Avsar-Ban, E., et al., Protein O-mannosylation is necessary for normal embryonic development in zebrafish. Glycobiology, 2010. 20(9): p. 1089-102. [165] Ohata, S., et al., Neuroepithelial cells require fucosylated glycans to guide the migration of vagus motor neuron progenitors in the developing zebrafish hindbrain. Development, 2009. 136(10): p. 1653-63.