SPARC is also concentrated circurnferentially at the apicolateral

142 Pages·2003·5.46 MB·English

SPARC is also concentrated circurnferentially at the apicolateral plasma membranes
of surface ectodermal cells. Injection of anti-sense Xenopus SPAPC morp;!olinos Ieads to
the dissociation of surface ectoderm by mid-tailbud. A similar dissociation is observed with
animal cap exp!ants taken fiom morpholino injected embryos. These studies indicate that
SPARC promotes epithelial cell-cell adhesion and is required for iaaintaining the integrity
of the embryonic epidermis of Xenopus embryos. This represents the first evidence of an
adhesive tiinction for SPARC.
Collectively, my studies indicate that SPARC tiinctions intracellularly as a calcium-
dependent regulator of ciliary movement and that SPARC has adhesive activity in epithelial
tissues, in contrast to an anti-adhesive activity in rnesenchymaI tissues. Since the
associations of SPARC with cilia and circumferentia! enrichment are observed in
amphibians and mammals, it is likely that these intracellular and extracellular fùnctions of
this glycoprotein are evoluticnarily conserved.
Table of Contenis
Table of Corztenîs iv
List of figures and Tables vii
Thesis Introduction
Review ùf ECM molecules
Review of SPARC structure and fùnction
Review of early Xenopus development
Xenoprrs as a mode1 organism
Objectives und Approach 18
Chapter 1 :A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm ce11
migration during Xenopus laevis gastrularion: eviùènce of counter-adhesive activity in
vivo
Abstract 2 1
Introduction 23
Materials and Methodr
Embryo Rearing
Microinjection
Histolo~y
Scanning Electron Microscopy
Whclemount In Situ Hybridization
Results
Peptide 4.2 is associated with an inhibition cf prospective head 3 1
mesoderm involution during gastrulation
Peptide 4.2 inhibits spreading of involuting prospective head 3 9
mesodermal and endodermal cells
Peptide 4.2 inhibits chordin expression in the anterior and 42
postenor region of tailbud embryos
Discussion 49
Chapter 2: Associalialionof SPARC (Osleonectin, BM-40)w ith extmcellrrlm and
infracellular components of the ciliated suflace ectoderm of Xenopus embryos
Abstract
Introduction
Materials and Methods
Embryos
Antibodies
Western Blot Analysis
Wholemount Immunocytochemistry
Animal Cap Assays and Northem Blot Analysis
Immunogold Electron Microscopy
Results
Mammalian anti-SPARC antibodies recognize epitopes on
Xet~opusS PARC
SPARC is present in the cilia of surface ciliated epidermal cells
Cell auton9mous activation of SPARC in the surface ectoderm
SPARC accumulates at the interface of surface epidermal cells
Sensorial layer cells express higher levels of SPARC mRNA
than the surface epidermal cells
SPARC mRNA expression in animal caps occurs in the
absence of exogenous factors
SPARC is associated with the ciliary microtubules
Discussion
Chapter 3: SPARC is associated with tubulin and promotes epithelial cell-cell adhesion
Abstract
Introduction
Materials and Methods
htibodies
Co-Immunoprecipitations
Whole-mount irnmunocytochemistry
Microinjection and Animal Cap Assays
Results
The CO-localizationo f SPARC and tubulin in cilia and neurons
Imrnunoprecipitation indicates a physiological interdction
between SPARC and tubu!in
SPARC CO-localizesw ith components of tight junctions
Decreases in SPARC translation lead to developmental
anomalies and dissociation of epidermal cells
Resctie of the morpholino phenotype with mouse and Xmopus
SPARC mRNA
Inhibition of SPARC mRNA translation by mcpholinos
Discussion
Thesis Discussion
ûverall Summary
References
Appendir A
List of figures and Tables
Figure 1. Schematic representation of the molecular organization 33
and biological activities of SPARC
Figure 2. Injection of low doses of peptide 4.2 and LiCl into the blastocoel 3 7
cavity of stage 8/9 embryos result in different rates of blastopore ring
closure and failure in development of anterior structures
Figure 3. Injection of peptide 4.2 results in incomplete gastrulation, with 4 1
embryos lacking anterior structures
Figure 4. Scanning electron microscopy indicates that peptide 4.2 inhibits 44
the spreading of cells at the leading edge of the involuting
chordomesoderm
Figure 5. Peptide 4.2-injected embryos show decreased chordin RNA 48
expression during gastrulation and in the chordoneural region of
tailbud embryos
Figure 6. Immunoblot of Xenoptcs and mouse tissues (whole lysates) 63
using a monoclonal antibody raised against human SPARC
(AON-503 1)
Figure 7. Scanning electron micrograph of the surface epidermis at 65
mid-tailbud (stage 35) showing evenly distributed ciliated cells in
the anterior region
Figure 8. Distribution of SPARC as revealed by imrnunocytochemistry 67
and in situ hyoridization
Figure 9. SPARC mRNA expression is activated in animal caps 7 1
Figure 10. Irnmunoelectron rnicroscopic analysis indicates that SPARC is 74
associated with the microtubules of axonemes
Figure 1 1. Co-localization of SPARC and tubulin in surface cilia and 9 1
neural tube
Figure 12. Tubulin is irnmunoprecipitated by anti-SPARC antibodies that 94
cross-react with Xenoptrs SPARC
Figure 13. Confocal serial sectioning indicates that SPARC is CO-localized 98
with zonula occludin- 1 at apicolateral plasma membranes of
epithelial cells
Figure 14. Inhibition of ectodermal ceIl adhesion by anti-sense SPARC IO 1
Morpholinos
Figure 15. Animal cap dissociation is rescued by SPARC sense cRNAs 1 04
Figure 16. Anti-sense Xenopus SPARC morpholinos inhibit SPARC 10 7
mRNA translation
TABLE 1: Defects in Xenopus Iaevis embryos associated with the
injection of SPARC peptide 4.2 in comparison tgLiC1.'
TABLE II: Rescue of XSMO-induced animal cap dissociation by
CO-injectiono f Xerroptcs or mouse SPARC sense cRNAs.
First and foremost, 1 am indebted to my supervisor Dr. Maurice Ringuette for his intellectual
guidance, support, enthusiasm, and genuine concem for my well-being and scientific growth
throughout my years in the laboratory. Reaching the destination was the ultimate goal, but I
owe him great thanks for making the journey such an important a.nd enjoyable part of the
trip.
1 would also Iike to thank members ofthe faculty: Drs. Ian Brown, Theodore Brown, UIli
Tepass, and Jaro Sodek for their technical and intellectual guidance.
1 also owe a great deal of gratitude to former and present members of the 6" floor. Firstly, 1
would like to thank Sashko Damjanovski for providing me with the basic molecular tools
and a foundation on which to start my graduate studies, and for his advice and suggestions
throughout my degree. To Vernadeth Alarcon, for her constant support and encouragement
during the writing of this thesis, and for providing an understanding ear and sound advice
when 1 was in dire need of it. To Yusuke Marikawa, for discussions and advice on
experiments, and for being a mode1 scientist. 1 have also been incredibly fortunate to have
had the companionship of many fellow students on the ' 6 floor, who provided the necessary
distractions and countless laughs and good times over the years. To CJ, Yvonne, Liz, Nat,
Magda, Derek. Michelle, Lea, and countless others over the years; you've made the lab a
second home, and at times in this degree, a tirst home. 1 appreciate your fnendship more
than words can adequately express, and will remember Our time together with great fondness
always.
1 also would like to acknowledge other fiiends who have provided stress-relief over the last
several years: Al, Nancy, Janani. Makeda, Michael, and Erica. Your understanding, words
of support, and willingness to listen when 1 needed it are much appreciated.
1 would like to thank rny family for allowing me the tieedom to pursue my goals, and for
understanding al1 that it involves. To my niece Vanessa, for coming over to play and for
reminding me of what's important in life. Lastly, I'd Iike to thank my "old" fi-iends, Yin-
Ling, Keren, Hoa, Kelly, Binh, Erika, and Sue. Tme friends are hard to find, and even
harder to hold onto, and 1 thank you for being there with me al1 these years.
Introduction
The extracellular matrix (ECM) of metazoans is composed of a complex network of
macromolecules with diverse morphoregulatory tiinctions. in addition to contributing to the
unique physiochernical properties and architecturai design of tissues, ECM molecules act as
potent regulators of many cellular activities. For exarnple, cell proliferation, differentiation,
migration, signal transduction, and survivai are dependent upon a dynamic reciprocal dialogue
between cells and the surroundhg ECM. Mutations or misexpression of ECM rnolecules is the
underlying cause of a broad range of morphological defects and diseases (Lukashev and Werb,
1998). This introduction will begin with a brief review of the four classes of ECM molecules,
and their structurai and regulatory contributions to development, pattern formation and tissue
remodeling. This is followed by a review of the properties and putative activities of SPARC, the
focus of my thesis. Lastly, a description of the key morphological events and developmental
stages of eariy Xenopus laevis d lbe given, providing criticai background into the experimental
design of my experiments and interpretation of my data. Special emphasis is placed on unique
aspects of Xetiopirs somitogenesis and embryonic skin development.
Structural and functional diversitv of ECM molecules
The €CM of animal cells is comprised of four major classes of macromolecules:
collagens, proteoglycans, elastins, and glycoproteins. Different combinations of ECM
rnacromolecules are co-assembled in different tissues, providing tremendous structural and
tiinctional diversity.
Collagens comprise the most abundant class of ECM molecules, accounting for up to
25% of the total protein content of some mammais. Collagen molecules include variable
amounts of a characteristic triple helicai structure comprised of three a chains wound around
each other into a right-handed superhelix. The a chains are constmcted of a repeating Glycine-
X-Y sequence motif, where X and Y can be any amino acid, but is often proline and
hydroxyproline respectively (Hay, 1991). Glycine plays a critical role in the tight folding of the
triple-heiix whereas hydroxyproiine-rnediates interchain H-bonding and ensures the assernbly of
a thermaily stable superheiii. Once secreted, collagens can form a complex variety of
supramolecular structures. Based on their design, collagens are divided into six classes: fibril-
forming (types 1, II, III, V, M), FACIT: fibril-associated with intempted triple helices (types
TX. XII), network-forming (IV), flamentous (VI), short chain (VIII, X), long chah (VII). Fibril
forming collagens are secreted as procollagens that require N- and C-terminal proteolytic
processing before they can self-assemble into striated fibrils. During fibrillogenesis, collagens
are stabilized by intramolecular and intermolecular covalent cross-links by lysyl oxidase at
terminai lysine residues (Kagan, 2000). The cross-linking generates fibrils with tremendous
tensile strength. In contrast to fibrillar collagens, network-forming collagen type IV molecules
are secreted as mature molecules and seiI-assemble in extended cornplex polygonal sheets. Type
IV collagen assernbly is stabilized by N-terminal tetramerization, C-terminal dimerization and
lateral zssociations. Lateral associations are possible because, unlike in type 1 collagen, the
collagenous dornains (triple-helical domains) of type IV collagen are intempted by numerotis
non-collagenous dornains. These two major classes illustrate how different a-chains lead to the
formation of radically different supramolecular structures. Moreover, different cornbinations of
collagens are ofien CO-assernbledw ith one another, playing a key role in determining the
biophysical properties of fibrils. For exarnple, the ratio of type 1 to type III collagen in tissues
is a prime determinant of tissue flexibility, such as in arteries (Intengan and Schifin, 2000).
SPARC has a strong affinity for both fibrillar collagens and type IV collagen (Yan and Sage,
1999). However, its role in the assernbly and fùnction of these collagens remains to be
detennined.
Profeo~Ivcars
-
Proteoglycans (PGs) are composed of glycosaminoglycan (GAG) chains attached to a
core protein. GAGs are long linear polymers of repeated disaccharides. Usually one sugar is D-
glucoronic acid or L-iduronic acids and the second sugar is either N-acetylglucosamine or N-
acetylgalactoseamine. GAGs faIl into three groups or types: chondroitiddermatan sulfate
(CSDS), keratan sulfate (KS), and heparan sulfàtc: (HS). Two features add to ttic tremendous
variety of PGs found in tissues. First, core proteins Vary in sequence and size, ranging From 10
KDa to 400,000 KDa (Lander AD, 1999). Moreover, the number, length, and type of GAG
chains associated with individual proteins Vary immensely. The tissue and cellular distribution
of individual PGs also Vary. While the majority of PGs are secreted, some are membrane-
spaming, linked to plasma membranes via phospholipids. or as in the case of serglycin, located
in storage vesicles inside cells (Kolset and Gallagher. 1990).
Through their high negative charge densities, the GAG chains associate with large
amounts of water molecules, aiid are prirnarily responsible for the viscoelastic properties of
tissues. In addition, PGs also regulate cell-cell and cell-matrix interactions. For example,
syndecan promotes the binding of integrins to matrix glycoproteins such as fibronectin (Woods
et al., 2000). Moreover, PGs can also act as reservoirs for growth factors and can present
growth factors to their cognate cell surface receptors. In some case, surh as fibroblast growth
factor, the growth factor cannot interact with its cognate receptor unless bound to a
proteogly~m( Botta et al., 2000).
Elastin
Elastin is the major protein of elastic fibres, and is responsible for imparting extensibility
and resiliency to tissues such as the dermis, ligaments, lung and major blood vessels (Mecham,