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Cloning, expression, and function of laminin in neuronal guidance Bonner, Jennifer 2001

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Cloning, expression, and function of laminin in neuronal guidance by Jennifer Bonner B.A., The University of New Hampshire, 1992 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS OF THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF GRADUATE STUDIES (Graduate Program i n Neuroscience) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A February 2001 ©Jennifer Bonner, 2001  In presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  publication of  and study.  thesis for scholarly by  this  his  or  her  Department The University of British Columbia Vancouver, Canada  requirements that the  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  DE-6 (2/88)  the  that  advanced  Library shall make it  by the  understood be  an  permission for extensive  granted  is  for  allowed  that without  head  of  my  copying  or  my written  Abstract The proper guidance of migrating growth cones during development relies on the balance of multiple guidance cues in the embryonic environment. In addition to guidance cues, growth cones are in contact with other substrates that may contribute to the pathfinding of neurons. For example, in the developing insect peripheral nervous system, pioneer neurons migrate on and between layers of the basal lamina, which consists of a network of many different proteins. Previous studies have demonstrated that one basal lamina molecule, laminin, promotes outgrowth of many classes of neurons in vitro. Laminin is a major component of the basal lamina and is a potent promoter of neurite outgrowth in vitro. In order to determine the role of laminin in neuronal development, grasshopper laminin subunit genes were cloned. Laminin was found to be expressed by hemocytes and laminin protein was abundant in the basal lamina throughout the embryo. Laminin expression was coincident with the outgrowth and guidance of the Tibial (Til) pioneer neurons in the developing limb bud. Laminin deposition in the basal lamina was conferred by hemocytes that migrate within the lumen of the limb with no apparent trajectory. In spite of this, laminin immunoreactivity in the basal lamina was uniform and is available as a substrate for axonal outgrowth. In this study, the simple grasshopper nervous system was used to investigate the role of laminin in neuronal pathfinding. The role of two sites on laminin was investigated using subunit specific antibodies and peptides as blocking reagents in vivo. While reagents aimed at a cell adhesion motif on the (3 chain did not affect T i l pathfinding, antibodies and peptides that block the nidogen binding site on laminin resulted in stalled Ti 1 axon migration, predominantly at the precise location in the trochanter, where they  ii  make a stereotyped ventral turn. After prolonged culturing, T i l axons remained stalled at the same location. The basal lamina in blocked embryos appeared to suffer no obvious structural defects, as assessed with immunofluorescence. Therefore, while T i l axons were capable of some outgrowth in the presence of blocking reagents, they were not able to navigate an essential turn and complete the trajectory to the CNS. This data suggests that the Ti growth cone's interaction with the nidogen binding site on laminin is vital for neuronal pathfinding in vivo.  iii  Table of Contents Abstract List of Figures List of Tables List of Abbreviations Acknowledgements Dedication C H A P T E R 1: B A C K G R O U N D Instructive vs. permissive cues Laminin and the basal lamina  ii vi vii viii x xi 1 1 2  Laminin Receptors 6 Role of laminin, laminin receptors, and other basal lamina molecules in maintaining basal lamina integrity The role of laminin in neuronal development: Implications from in vitro studies Genetic analysis of laminin function The T i l pioneer neuron pathway C H A P T E R 2: C L O N I N G A N D EXPRESSION OF GRASSHOPPER L A M L N I N Introduction Methods Cloning of Laminin (3 and y chains in Schistocerca gregaria Northern Analysis In situ hybridization  9 11 11 15 18 26 26 30 30 32 32  Generation of Fusion Protein Constructs  33  Antibody Staining  34  Pre-absorption of sera with fusion protein  34  Confocal Microscopy  35  Time Lapse Nomarski Microscopy  35  Results  36  Laminin family members in Schistocerca gregaria  36  Expression of laminin in the developing embryo  40  Laminin is expressed by hemocytes  47  Embryonic hemocytes migrate randomly in the developing grasshopper limb  58  Discussion Laminins in grasshopper  65 65  Embryonic hemocytes migrate randomly in the limb bud and secrete components of the basal lamina  67  Establishment of even laminin expression by migratory hemocytes  68  iv  C H A P T E R 3: L A M I N I N IS REQUIRED FOR T i l A X O N G U I D A N C E  71  Introduction  71  Methods Antibody Staining  75 75  Pre-absorption of sera with fusion protein  75  Confocal Microscopy  76  IgG purification Dialysis Western analysis of fusion proteins Western analysis of embryonic lysate Culturing  76 77 77 77 78  Blocking peptides  78  Blocking antibodies Assessment of Basal Lamina integrity in blocked embryos  79 79  Results 79 Localization of laminin receptors on T i l growth cones and axons 81 Blocking a conserved nidogen recognition sequence on laminin disrupts axon guidance 86 T i l growth cones remain stalled after prolonged culturing 91 T i l axons stall within filopodial range of guidance cues found within the trochanter 92 Basal lamina integrity Blocking Beta chain function has no effect on T i l pathfinding Discussion  93 107 112  Potential roles of nidogen The role of basal lamina in neuronal pathfinding  112 114  Growth cone priming is essential for pathfinding  115  C H A P T E R 4: S U M M A R Y A N D CONCLUSION Future studies  121 125  Nidogen localization  126  Integrins  126  LAR  127  Concluding remarks References  128 129  v  List of Figures Figure 1: The laminin heterotrimer 5 Figure 2: Laminin interacts with other basal lamina molecules as well as receptors 8 Figure 3: Illustration of the developing limb bud at approximately 34% of development. 21 Figure 4: Alignment of grasshopper laminin deduced amino acid sequences with mouse and Drosophila laminin 39 Figure 5: Expression of laminin in the developing grasshopper embryo 42 Figure 6: Immunofluorescence of laminin in the CNS and the limb bud 46 Figure 7: Glial cells do not express laminin... 50 Figure 8: Laminin is expressed by a subset of mesodermal cells that are distinct from muscle precursors 53 Figure 9: Laminin is expressed by hemocytes 55 Figure 10: Migratory hemocytes express laminin 62 Figure 11: Several migrating cells in the limb bud express laminin 64 Figure 12: Time course of laminin expression in relation to the T i l pioneer neuron pathway 83 Figure 13: Laminin is expressed by hemocytes at 30% and 35%> of embryonic development in the grasshopper limb bud 85 Figure 14: (31 integrin is expressed in the developing limb bud 90 Figure 15: Schematic illustration of types of T i l pathfinding errors observed in cultured embryos 95 Figure 16: Disrupting a conserved nidogen recognition sequence results in aberrant neuronal pathfinding 97 Figure 17: Summary of laminin antibody and peptide blocking experiments on the y chain 99 Figure 18: Schematic depicting the three possible locations of T i l stalled axons when laminin is blocked 102 Figure 19. Ti axons stall in a discrete location in the limb bud when laminin is blocked. 104 Figure 20: Basal lamina and secreted guidance cue localization are intact in lamininnidogen blocked embryos 106 Figure 21: Summary of laminin antibody and peptide blocking experiments on the (3 chain 109 Figure 22: Summary of results of different doses of antibodies and peptides designed to disrupt laminin (3 chain function Ill Figure 23: Proposed mechanism of laminin in axon guidance 120  vi  List of Tables Table 1: Summary of immunofluorescent results 57 Table 2: List of migrating cells that were characterized based on distance traveled and velocity 60  List of Abbreviations bp  basepairs  BCIP  5-bromo-4-chloro-3-indolyl-phosphate  BL  basal lamina  Ca  2 +  calcium  CaCh  calcium chloride  Cx  coxa limb segment  Cy3  indocarbocyanine  DPNAV  aspartate-proline-asparagine-alanine-valine  ECM  extracellular matrix  EGF  epidermal growth factor  EL  epithelial layer  Fe  femur limb segment  FITC  fluorescein isothiocyanate  GST  glutathione s-transferase  HPvP  horse radish peroxidase  kb  kilobase  kDa  kilodalton  MP  median precursor  mg  milligram  MgCb  magnesium chloride  MgS04  magnesium sulfate  ml  milliliter  viii  mM  millimolar  NBT  4-nitro blue tetrazolium chloride  ng  nanogram  PANDV  proline-alanine-asparagine-aspartate-valine  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PMSF  phenylmethylsulfonyl fluoride  PNS  peripheral nervous system  RT-PCR  reverse-transcriptase polymerase chain reaction  SDS  sodium dodecyl sulfate  SYGAR  serine-tyrosine-glycine-alanine-arginine  Ti  tibia limb segment  Tr  trochanter limb segment  Lig  microgram  jul  microliter  LIM  micromolar  YAGSR  tyrosine-alanine-glycine-serine-arginine  ix  Acknowledgements I would like to thank my supervisor Tim O'Connor for fostering an independent learning environment and for supporting me throughout the duration of this project. It's been a lot of fun working in his lab, and I have learned a lot. Also many thanks go out to past and present members of the O'Connor lab for creating a fun and supportive lab atmosphere, especially to Carolyn Isbister, Arthur Legg, and Colleen Wu. I would also like to thank Vanessa Auld, not only for being an integral member of my committee, but also for allowing me to conduct the bulk of the molecular characterization under her guidance in her lab. I would also like to thank my committee members, Cal Roskelley, Terry Snutch, and Wolfram Tetzlaff for providing technical and theoretical guidance over the years. I also gratefully acknowledge the following individuals for the generous gift of reagents and expertise that were essential to this study: Eldon Ball for the mes-3, 3H12 and 15 antibodies, Sarb Ner for the anti-repo antibodies, Michael Bastiani and Kai Zinn for cDNA libraries, Mark Seeger for primers, Sal Carbonetto for (31 integrin antibodies, K i m Gerrow for providing limb fillets and Ken Norman for confocal expertise.  x  Dedication I found it difficult to decide on one person to dedicate this thesis to, since I have drawn on many people in my life in order to complete the goals that I set out for myself. There are many that have been scientific mentors, but this thesis is dedicated to those people whose influence went beyond science. This thesis is dedicated to my mom, who for my entire life has taught me by example that hard work, tenacity, intelligence and strength of character can overcome any adversity. This is also dedicated in loving memory to Dorothy, whose faith in me will stay with me forever. Finally, this thesis dedicated to Ken, for love, support, laughter, and for sharing with me this adventure and many others.  xi  CHAPTER 1: BACKGROUND Instructive vs. permissive cues During development of the nervous system, axons extend along many different types of substrates, including other neurons, glia, epithelium, muscle, and basal lamina. Whereas the variety of potential sources of guidance information in the embryonic environment is extensive, the balance of molecular cues from these sources is highly orchestrated such that few errors are made during neuronal pathfinding. Not only is the generation and arrangement of appropriate guidance cues essential for correct pathfinding, so is the nature of the response of the motile tip of the axon upon detection of these cues. The growth cone, an actin rich, highly dynamic sensory/motor apparatus that recognizes and integrates guidance information, characterizes the tip of the migrating neuron. This information is translated into a reorganization of the actin and microtubule cytoskeleton, generating the appropriate steering response. Instructive guidance cues can be defined as molecules that, when discretely localized, instruct or guide neurons towards or away from a region. Instructive cues can be attractive or repulsive, and growth cones exhibit stereotyped and contrasting behaviors when contacting localized concentrations of either attractive or repulsive guidance cues. With the cloning of several families of guidance molecules, molecular counterparts of attractive and inhibitory guidance molecules have been identified, which comprise several classes of instructive cues. Whereas guidance molecules are typically categorized as either attractive or inhibitory, their effect on growth cone extension is not fixed, but  1  can be altered by various factors, such as the presence of other guidance molecules (Chen et al., 1998; Giger et al., 1998; Takahashi et al., 1998; Winberg et al., 1998), expression of different receptor subtypes on the growth cone (Hamelin et al., 1993; Bashaw et al., 1999; Hong et a l , 1999; Takahashi et al., 1999; Tamagnone et al., 1999), intracellular signaling cascades (Ming et al., 1997; Song et al., 1998), cleavage of guidance molecules, (Hattori et a l , 2000; Galko and Tessier-Lavigne, 2000) and the presence of basal lamina molecules (Hopker et al., 1999). Since the embryonic environment is molecularly complex and contains multiple cues, it is not surprising that growth cone guidance is more complex than can be explained by cues that confer either attractive or inhibitory information to the growth cone. A surveying growth cone is exposed to information from other sources in addition to instructive guidance cues. These cues are permissive, are often characterized by uniform expression, and are thought to facilitate neuronal outgrowth. With the complex and dynamic response of the growth cone to its environs, it is difficult to assess the role of each of these components independently. However, growth cones are not encountering cues in isolation, but as part of a complex molecular landscape. Therefore, understanding the nature of the interactions between instructive and permissive cues is essential to understanding growth cone guidance. One permissive cue that is important for neuronal outgrowth is laminin, which is abundant in basal lamina during neuronal development.  Laminin and the basal lamina Laminin is a heterotrimer, consisting of three distinct subunits, a, (3, and Y- Each subunit has a similar domain structure of heptad-repeat containing domains I and II,  2  followed by EGF repeat containing domain III a globular domain IV, another EGF repeat containing domain V , and a final globular domain VI (figure 1). This general structure has a few subunit specific modifications: the a chain contains a globular domain adjacent to domain I, as well as duplications of domains III and IV. The P chain has an a domain between domains I and II. Also, several variant chains of a, P, and yhave been cloned that have truncations in various domains (Colognato and Yurchenco, 2000). The mature laminin molecule has a cruciform structure characterized by long arms and short arms (figure 1). Domains I and II of all three subunits come together to form a coiled-coil which is strengthened by disulfide bonding. A l l laminin isoforms examined to date contain one each of the three subunits. The basal lamina is a thin extracellular array that separates tissue types, withstands tension, acts as a molecular sieve for secreted molecules and is a scaffold for migrating cells during embryonic development. A complex network of several molecules, the basal lamina includes type IV collagen, perlecan, nidogen, and laminin (Timpl and Brown, 1996). Not comprised of a random collection of molecules, the structure of the basal lamina relies upon highly ordered physical interactions of individual components that are required in order to serve the functions described above. Laminin binds to a number of proteins in the basal lamina, such as perlecan and nidogen (figure 2; Timpl and Brown, 1996). Importantly, laminin binds to itself, resulting in laminin polymerization which is Ca++ and concentration dependent (Yurchenco et al., 1985; Cheng et al., 1997). Type IV collagen also self-assembles, forming a collagen network (Yurchenco and Furthmayr, 1984). The laminin and collagen networks are spatially distinct in the basal lamina, however, linker molecules such as perlecan and nidogen  3  Figure 1: The laminin heterotrimer. Laminin has three subunits, a, (3, and y. A l l three subunits are assembled into a cruciform stucture that is mediated by a coiled-coil that is composed of domains I and II. For descriptions of domain structure, see text  4  5  connect the two networks. Within the context of the basal lamina, laminin exists as a heterotrimer within a laminin network that associates with many other molecules, suggesting a complex conformation that may have functional ramifications.  Laminin Receptors Due to the functional diversity of laminin, it is not surprising that many different receptor types transmit laminin signals. One family of laminin receptors is the integrin family. Integrin receptors are a family of E C M binding molecules involved in cell adhesion and migration. Integrins are heterodimers, consisting of a and (3 subunits. Six different ap integrin heterodimers bind to several isoforms of laminin (figure 2; Colognato and Yurchenco, 2000). Integrin activation by laminin can be modulated and several examples exist of differential activation of integrins in response to changes in laminin concentration and developmental age (see below). Non-Integrin receptors are equally important in mediating laminin function. pi,4 galactosyltransferase binds laminin and galactosylates it, and this galactosylation is required for laminin induced PC 12 neurite outgrowth in vitro (Begovac et al., 1991; Begovac et al., 1994; Begovac and Shur, 1990). LBP-110 binds the neurite outgrowthpromoting domain of the a chain, I K V A V (figure 2), and mediates neuronal outgrowth (Kibbey et a l , 1995; Powell and Kleinman, 1997). The 67 kD receptor binds to the cell adhesion motif, YIGSR, on the p chain. The 67 kD receptor is thought to modulate integrin binding to laminin, (Graf et al., 1987; Powell and Kleinman, 1997; Menard et al., 1997). Interacting with the globular domain of the a chain of laminin, a-dystroglycan is  6  Figure 2: Laminin interacts with other basal lamina molecules as well as receptors. Illustrated schematically are some sites of interactions between laminin and its basal lamina and receptor partners. Receptors are in circles, basal lamina molecules are in boxes. Also indicated are functionally relevant peptide motifs (plain text) YIGSR ( Y A G S R in grasshopper), D P N A V , and I K V A V . YIGSR is a cell adhesion motif that is involved in netrin signaling, D P N A V binds nidogen, and I K V A V elicits neurite outgrowth in vitro. Listed are the various players that have been implicated in neuronal growth and guidance or basal lamina integrity. See text for details.  7  a laminin receptor found at the vertebrate neuromuscular junction on the post-synaptic cell (Ervasti and Campbell, 1993; Patton et al., 1997; Colognato and Yurchenco, 2000). Now emerging as a major player in neuromuscular junction development, laminin has been shown to cluster both acetylcholine receptors and a-dystroglycan which associates with the cytoskeleton in muscle (Sugiyama et al., 1997; Cohen et al., 1997; Montanaro et al., 1998). Finally, the L A R family of receptor tyrosine phosphatases interacts with laminin nidogen complexes and mediates HeLa cell spreading on a laminin-nidogen substrate (O'Grady et al., 1998).  Role of laminin, laminin receptors, and other basal lamina molecules in maintaining basal lamina integrity Self-assembled type IV collagen and laminin networks are connected to one another through interactions with other molecules such as nidogen and perlecan. However, in vivo, basal lamina molecules such as laminin are not necessarily homogeneous in distribution. For example at the neuromuscular junction, laminins are specifically distributed along the synapse and are developmentally regulated. There are at least five isoforms of laminin expressed at the neuromuscular junction (Patton et al., 1997). Mutations in the (32 chain of laminin (enriched in the synaptic basal lamina) results in defects in synaptic development, in spite of molecular compensation by other laminin subunits, indicating the functional specificity of laminin isoforms (Noakes et al., 1995a; Patton et al., 1997; Patton et al., 1998). The appropriate localization of a given basal lamina molecule relies on proper expression and secretion, binding to basal lamina constituents (including self) and binding to cell surface receptors. This dynamic interplay  9  between basal lamina constituents and receptors ensures both the structural integrity of the basal lamina and the proper spatial distribution of basal lamina components. Genetic analysis of basal lamina components has implicated some, but not all, in basal lamina integrity. Laminin, type IV collagen, and perlecan are crucial in maintaining the structural integrity of the basal lamina (Garcia-Alonso et al., 1996; Costell et al., 1999; Smyth et al., 1999; Miner and L i , 2000; Norman and Moerman, 2000). Interestingly, in the case of perlecan and type IV collagen mutants, basal lamina assembly is unaffected, but under mechanical stress, the mutant basal lamina cannot withstand tension and ruptures (Costell et al., 1999; Norman and Moerman, 2000). The basal lamina in Drosophila larvae mutant for the a chain of laminin exhibit brakes at the site of axonal contact (Garcia-Alonso et al., 1996). In mouse, a mutation in the a5 chain results in a defective glomerular basement membrane. Furthermore, y\ laminin mutant mouse embryos and embryoid bodies are lacking the basal lamina (Smyth et al., 1999). Surprisingly, nidogen, although serving as a physical linkage between type IV collagen and laminin networks, is not essential for basal lamina stability (Kim and Wadsworth, 2000; Kang and Kramer, 2000; Murshed et a l , 2000). Receptors for basal lamina molecules have also been implicated in basal lamina stability. Among laminin receptors are the integrins and dystroglycan which bind to different motifs in laminin (figure 2). Although mice mutant for integrin subunits die early in embryogenesis, analyses of mutant embryos and embryoid bodies have demonstrated the importance of integrin for basal lamina assembly (Stephens et al., 1995; Bloch et al., 1997; D i Persio et al., 1997; Sasaki et al., 1998; Aumailley et al., 2000). Dystroglycan, which binds to the carboxy tail of the a chain (figure 2) was found to not  10  only be essential for basal lamina integrity, but is also integral in laminin network polymerization (Williamson et al., 1997; Henry and Campbell, 1998; Colognato et al., 1999; reviewed by Colognato and Yurchenco, 2000). Thus the appropriate integration of basal lamina molecules requires the coordinated function of integrins, dystroglycan, laminins, type IV collagen, and perlecan.  The role of laminin in neuronal development: Implications from in vitro studies Over two decades ago it was shown that conditioned medium from various tissue types contained a neurite outgrowth promoting activity. One of these activities was identified as laminin by biochemical purification and antibody studies (Collins, 1978; Lander et al., 1985). Since the discovery of its potent neurite outgrowth promoting activity, laminin has been used as a substrate for neurite growth in vitro. A l l laminin isoforms have three subunits, (a, (3 and y) that assemble into a cruciform structure. In vertebrates, there are presently 5 a, 3 p\ and 3 y chains that comprise 12 isoforms of laminin, each containing a distinct combination of the three subunits (Patton et al., 1997; Colognato and Yurchenco, 2000). The different laminin isoforms typically exhibit different expression patterns and functions (Hunter et al., 1989; Noakes et al., 1995a; Lentz et al., 1997; Patton et al., 1997). Functional domains in each subunit that promote neurite outgrowth, cell adhesion, cell migration, receptor binding, and E C M molecule binding have been described (figure 2, Graf et al., 1987; Tashiro et al., 1991; Calof et al., 1994; Nomizu et al., 1996; reviewed by Powell and Kleinman, 1997).  11  Laminin mediated axon outgrowth in vitro has been a useful paradigm for examining the mechanisms of extension of axons. For example, when presented with a stabilized source of laminin conjugated to a bead, dorsal root ganglion neurons turn toward the laminin coated bead. A n increased velocity of axonal extension follows this redirected growth (Kuhn et al., 1995). The laminin induced turning response is abolished by protein kinase C (PKC) inhibitors and requires extracellular calcium whereas the increased axonal velocity is dependent on Cam Kinase II (Kuhn et al., 1998). In subsequent analyses, the stimulus history of a neuron was implicated as an important modulator of growth cone guidance. Using the same paradigm, Diefenbach et al. (2000) demonstrated that the growth cone response to a laminin coated bead depended on the previous experience of the growth cone. For example, initial encounters with laminin coated beads caused a stereotypical increase in axon extension, while subsequent encounters elicited a market different response, namely, the growth cone stalled. These results shed light on the possible mechanisms of permissive cues. Perhaps sampling laminin changes the molecular componentry within the growth cone, which alters the response to subsequent molecular encounters, whether similar or dissimilar. Neurons will reorient their growth in response to a stabilized source of laminin, but can they detect and respond to gradients of laminin? McKenna and Raper (1988) demonstrated that chick sympathetic ganglion neurons exhibit no preference to a gradient of laminin. The axons did not reorient their growth to grow up the concentration gradient, and mostly grew straight until the laminin concentration was low enough to cease growth. Subsequent work indicated that another class of neurons, retinal neurons, do reorient their growth when presented with a gradient of laminin -2 (Halfter, 1996). It  12  is difficult to compare these two experiments directly because quantification of the slope of the gradients differs significantly. Additionally, different isoforms of laminin were used in each study. However, these results suggest that when presented alone in vitro, laminin may have an instructive role in neuronal guidance. Although these in vitro results provide insight into the mechanism of laminin guidance in vivo, they may not reflect the function of laminin within the complex basal lamina. During development of the retino-tectal tract, embryonic retinal cells extend axons along the optic tract, which is rich in laminin. In vitro, embryonic day 6 retinal cells attach and extend neurites on laminin by an integrin-dependent mechanism, whereas older cells do not respond to laminin (Cohen et al., 1986; Hall et al., 1987). The age at which embryonic retinal cells stop adhering and extending processes on laminin in vitro , corresponds to the time when the axons have reached the tectum (their target) in vivo. If the tectum is ablated, the responsiveness to laminin is retained in older cells, indicating a signal originating from the tectum is rendering the cells unresponsive to laminin (Cohen et al., 1989). The loss of responsiveness of retinal cells can be accounted for by down regulation oc6 integrin mRNA and protein as well as by desensitization of integrin receptors as described with platelets, leukocytes and other cells (Hall et al., 1987; de Curtis et al., 1991; Hynes, 1992). The inability of retinal neurons to extend on laminin can be rescued by conformational changes to the laminin molecule, including proteolytic cleavage of laminin (Calof et al., 1994) and the addition of antibodies that react with the domain Vl-IVb of the a chain (Ivins et al., 1998). Furthermore, retinal growth on laminin can be restored by extracellular or intracellular activation of integrins indicating  13  that down-stream mechanisms to integrin binding laminin are in place in non-responsive neurons (Ivins et al., 2000). Condic and Letourneau (1997) have also shown differential regulation of integrin receptors in response to changes in laminin concentration in vitro. Cells cultured overnight on low concentrations of laminin express higher surface levels of the integrin a6 receptor as compared to cells cultured on 10 fold more laminin or fibronectin. This increased availability of integrin a6 receptor at the surface could potentially allow axons to continue a fast rate of extension even in the absence of high levels of laminin substrate. This is one mechanism by which a balance of adhesion to the substratum and motility of cells could arise. There are several processes identified that may be important for neurons to extend axons on laminin. Firstly, laminin must be properly distributed in the basal lamina, which is key to laminin function, for example at the synapse (Patton et al., 1997), where certain laminin isoforms, which are discretely localized, prevent glial cell invasion into the synaptic cleft (Patton et al., 1998). In addition, laminin polymerization into polymeric complexes (which could be important for the functions of laminin, for example absorbing mechanical stress) could be important for axonal extension. Laminin conformation may be further altered by metalloprotease cleavage of laminin isoforms. Epithelial cells that normally avoid migrating on laminin-5 will migrate onto laminin-5 substrates in the presence of a secreted metalloprotease (Giannelli et al., 1997). Laminin can interact with neurons through many receptor types, but the best described are the integrin receptors. Integrin affinity for laminin can be modulated, which is likely to result from a conformational change in laminin (Ivins et al., 1998), and  14  can be overcome by intracellular or extracellular activation of integrins (Ivins et al., 2000). Laminin induced growth cone turning, relies on P K C and C a al., 1998). C a  2+  2+  influx (Kuhn et  is sufficient to mediate growth cone turning, and is downstream of netrin  signaling, suggesting that C a is a common mediator of growth cone behavior (Hong et 2+  al., 2000; Zheng, 2000). Laminin signaling that induces neuronal growth must ultimately converge on actin, the major cytoskeletal element in neuronal growth cones. Activators of actin polymerization are downstream of receptors and include the rho family of GTPases, which are activated by guanine nucleotide exchange factors (GEF) such as trio (Bateman, et al., 2000). Rho GTPases, such as Cdc-42 polymerize actin through N - W A S P and Arp2/3 complexes, which bind directly to actin. N - W A S P appears to have both an actin severing and an actin polymerization activity (Korey and Van Vactor, 2000). In another (but not exclusive) pathway, the phosphorylation state of enabled (which is a substrate for receptor tyrosine phosphatase, D-lar, and the tyrosine kinase Abl) is important for directing actin assembly (Korey and Van Vactor, 2000). Although these components have not been directly linked to laminin induced neurite outgrowth of guidance, they are emerging as common mediators of growth cone responsiveness.  Genetic analysis of laminin function A genetic approach has been useful to examine the potential roles of laminin in nervous system development, including axon guidance. Mutations in the laminin a chain in Drosophila and the a chain in C. elegans have been isolated. A hypomorphic a chain mutation in Drosophila results in defects in the pathfinding of a sensory pioneer neuron (ocellar neuron) (Garcia-Alonso et al., 1996). Mutations in the laminin-binding PS1 15  integrins in Drosophila result in non-specific pathfinding defects, suggesting integrinlaminin may be involved in the modulation of guidance responses (Hoang and Chiba, 1998). In C. elegans, mutations in the a chain of laminin cause motoneuron axon defects including dorsal and ventral migrations and increased fasciculation defects (Forrester and Garriga, 1997). Defasciculated axons are also prevelant in mutants for the a integrin subunit (Baum and Garriga, 1997). In vertebrates, laminin containing the (32 subunit is found in active zones at the neuromuscular junction, and marks sites of reinnervation on the muscle after injury. (Sanes and Hall, 1979; Hunter et al., 1989). The neuromuscular junction of mice deficient in the (32 subunit of laminin exhibit abnormal nerve terminal morphology, and a decreased number of presynaptic active zones; they lack localization of presynaptic vesicles, and exhibit a decreased spontaneous release of neurotransmitter at affected synapses (Noakes et al., 1995a). In addition, (32 isoforms of laminin are crucial in maintaining the close opposition of nerve terminal and the muscle by preventing glial cell invasion into the synaptic cleft (Patton et al., 1998).  Analysis of |32 containing  laminin isoforms in the central nervous system reveals that these synaptic laminins play analogous roles at peripheral and central synapses. Retinal synapses of (32 mutant mice have an immature morphology, including presynaptic active zones that are separated from the post-synaptic membrane by gaps. This indicates that (32 containing laminins found in the synaptic basal lamina may be the synaptic linkage between pre-and postsynaptic membranes. Interestingly, a (32-containing laminin was biochemically purified from synaptosomes in a pre-synaptic complex that included a calcium channel and nonerythroid spectrin (Sunderland et al., 2000). These sites of neurotransmitter release could  16  be coupled to their post-synaptic partners through (32 laminin isoforms and the laminin receptor oc-dystroglycan. a-dystroglycan is localized to acetylcholine receptor hot spots and thus is co-localized with (32-containing laminins (Hunter et al., 1989). Due to the diverse functions of laminin, these genetic analyses are not without caveats. First, the stability of the laminin heterotrimer relies on the presence of all three subunits (Matsui et al., 1995), making the analysis of single subunits difficult. In addition, these genetic analyses have uncovered the requirement of laminin for the stability and maintenance of the basal lamina as mentioned earlier. In Drosophila, defects in sensory pioneer pathfinding are accompanied by breaks in the surrounding basal lamina (Garcia-Alonso et al., 1996), which could result in secondary effects such as mislocalization of secreted guidance molecules such as netrins and semaphorins. In addition to basal lamina disruptions in the mouse laminin mutants, compensation for the loss of the subunits occurs by regulation of other subunits (Noakes et al., 1995b; Patton et a l , 1997). An additional caveat is the requirement of laminin in other developmental processes. Non-neuronal defects resulting from laminin mutations in Drosophila include heart and gut developmental abnormalities as well as myotube formation and muscle attachment defects (Yarnitzky and Volk, 1995). C. elegans laminin mutants exhibit several cell migration abnormalities including neuronal and muscle cell migration defects (Forrester and Garriga, 1997). Mice deficient in the laminin (32 subunit suffer kidney developmental abnormalities (Noakes et al., 1995b; Miner and L i , 2000) and mice lacking the y l chain of laminin die as embryos (Smyth et al., 1999). Thus genetic manipulation of laminin does not produce a clear picture of laminin function in neuronal  17  guidance due to the requirement of a single subunit for stability of the basal lamina, the aberrant expression of alternative subunits in mouse, and the obvious requirement of laminin in other developmental processes. This thesis work intended to overcome these experimental difficulties by using a cellular approach to test the function of laminin in vivo.  The TH pioneer neuron pathway The development of the grasshopper peripheral nervous system is ideally suited for the functional analysis of molecular guidance cues. As grasshopper embryos can be cultured for a period of days, a major attribute of this system is the ease with which embryos can be manipulated at precise developmental stages. Therefore, axon guidance mechanisms can be analyzed in a physiologically relevant context. One major nerve pathway that has proved extensively useful in the analysis and examination of growth cone steering and guidance is the developing Tibial (Til) pioneer pathway. In the developing limb bud, many of the mechanisms and molecules that mediate the guidance of the T i l pioneer sensory neuron projection have been described (Bate, 1976; Keshishian and Bentley, 1983; Caudy and Bentley, 1986a, 1986b, 1987; Condic and Bentley, 1989a, 1989b, 1989c; Bentley and O'Connor, 1992; Kolodkin et a l , 1992; Isbister et al., 1999; Isbister and O'Connor, 1999; Wong et al., 1999). The T i l cell bodies delaminate from the epithelium at approximately 30% of development and extend axons proximally toward the central nervous system (CNS). As the T i l growth cones migrate they make contact with several substrates including the laminin rich basal lamina (Anderson and Tucker 1989, Chapter 2), epithelium and several intermediate neuronal targets (Keshishian and Bentley 1983; O'Connor 1999). The T i l growth cones initially  18  extend proximally along the epithelium until they contact a pre-axonogenesis neuron, the T r l cell. Upon contact with the Trl cell located in the trochanter segmental epithelium, the T i l axons make an abrupt ventral turn (figure 3). Near the ventral midline of the limb, the growth cones contact another intermediate target, the C x i neurons, and turn proximally extending into the CNS (Keshishian and Bentley 1983; Bentley and O'Connor 1992). Contact with the C x i cell is essential for T i l axons to turn proximally and enter the CNS (Bentley and Caudy, 1983). Many neurons in the developing limb bud fasciculate with the T i l pioneer projection and use it as a scaffold to gain entry into the CNS (Klose and Bentley, 1989; Wong et al., 1997; O'Connor, 1999). For example, axons from the subgenual sensory organ (SGO) that arise distal to the T i l neurons extend along the epithelium until contact is made with the T i l neurons. Ablation of the T i l neurons results in a cessation of proximal growth of the SGO neurons (Klose and Bentley, 1989; Wong et al., 1997) demonstrating the importance of the T i l pathway.  Semaphorins guide TH neurons The semaphorins are a large family of guidance molecules and have been implicated in inhibition of neuronal outgrowth. This family of proteins can be subdivided into at least eight classes based on their structure and origin of isolation such as vertebrate, invertebrate and viral (Semaphorin Nomenclature Committee, 1999). Semaphorins are either secreted, or membrane associated through a transmembrane domain or a glycosylphosphatidylinositol (GPI) linkage. The T i l pathway is guided in part by members of the semaphorin family. In the developing grasshopper limb bud, two classes of semaphorins are found in distinct, relatively non-overlapping expression patterns. Grasshopper Sema-2a is expressed in a distal to proximal and dorsal to ventral  19  Figure 3: Illustration of the developing limb bud at approximately 34% of development. The Ti 1 pathway makes a stereotypic path to the CNS that consists of an initial proximal extension of T i l axons, followed by a series of turns that are emphasized in the text. The axons encounter several intermediate neuronal targets, including the F e l cell, the T r l cell, and the C x i cells. Transmembrane (tan) and secreted (pink) semaphorins are expressed during T i l growth and influence their path. The names of limb segments have been abbreviated as follows: Ti, tibia; Fe, femur; Tr, trochanter; and Cx, coxa.  20  Sema la Senna 2a  0  gradient in the epithelium at the time that the T i l pathway is generated (figure 3, Isbister et al., 1999). The dorsal and distal-most epithelium, that highly expresses Sema-2a, is avoided by the migrating T i l axons suggesting a repulsive activity for this secreted semaphorin. In the presence of blocking antibodies, T i l axons explored limb epithelium that expresses high levels of Sema-2a, areas they usually avoid (Isbister et al., 1999). Thus, similar to its vertebrate counterparts, secreted Sema-2a appears to confer repulsive information to a developing projection in the PNS. These results indicate that the distalproximal and dorsal-ventral gradient of Sema-2a plays an important role in directing the T i l growth cones proximally along the limb epithelium and possibly ventrally along the trochanter. Whereas Sema-2a was found to function as a repulsive guidance cue for the T i l growth cones, an alternative role for a transmembrane semaphorin (Sema-la) was described. Sema-la was initially found to have a role in guiding the T i l growth cones in the developing limb bud, however its mechanism of action was unclear (Kolodkin et al., 1992). It is expressed along circumferential bands of epithelium in the developing limb bud, most notably in the trochanter segment where the T i l pioneer neurons make the ventral turn (Kolodkin et al., 1992). Later in development, Sema-la expression arises along circumferential epithelial bands in the proximal tibia (just proximal to the SGO neurons) and in the proximal tarsus (Singer et al., 1995; Wong et al., 1997; Isbister et al., 1999). Although initial experiments clearly demonstrated that Sema-la was a guidance cue, whether it was attractive or repulsive was difficult to interpret. Blocking of Sema-la  22  with a monoclonal antibody resulted in marked defasciculation and axon branching of T i l axons in the region of the trochanter epithelium (Kolodkin et al., 1992). Normally the T i l neurons cease proximal growth within the Sema-la expressing epithelium, turn ventrally, and migrate along the Sema-la expressing band of epithelium. This behavior of the T i l axons within the Sema-la expressing epithelium is suggestive of an adhesive or attractive role for this molecule. Furthermore, this region of Sema-la expressing epithelium is more adhesive for T i l growth cones compared to other regions (Condic and Bentley, 1989b; Isbister and O'Connor, 1999). Also, T i l growth cones leave the Semala epithelium only after contact with a neuronal guidepost cell, the C x i cell, which has been shown to be essential for the proximal turn of the T i l neurons toward the CNS (Bentley and Caudy, 1983). By conferring adhesive or attractive information to the T i l axons, Sema-la ensures that they remain within the trochanter, which guarantees contact with the next essential cue, the C x i cell. The original antibody perturbation experiments support this role as blocking of Sema-la resulted in T i l growth cones extending aberrantly past the trochanter (Kolodkin etal., 1992). Alternatively, the results could also support an inhibitory model as antibody perturbation caused the T i l neurons to generate multiple non-fasciculated axon branches in the vicinity of the trochanter epithelium (Kolodkin et al., 1992). These results would be consistent with Sema-la acting as a repulsive cue, facilitating axon fasciculation and inhibiting axon sprouting. To clarify this, ectopic recombinant Sema-la was presented to T i l axons as they extended proximally along the femur epithelium prior to contact with the T r l cell. When the T i l growth cones contacted cells expressing Sema-la, the axons consistently abandoned their normal pathway and turned toward the Sema-la expressing cells (Wong  23  et al., 1999). Thus in the context of the variety of guidance cues sampled by the T i l neurons in the developing limb bud, high expression of Sema-la in individual cells was sufficient to mediate an attractive turning response (Wong et al., 1999). In this study, we set out to determine the role of laminin in the guidance of pioneer neurons. We hypothesized that laminin is evolutionarily conserved at the molecular level and would be abundantly disributed in basal lamina. In addition, laminin is likely to play a crucial role in axon pathfinding. By analyzing laminin function in grasshopper, we can determine the precise role of distinct sites on the laminin heterotrimer in axon guidance, without disturbing the milieu of the basal lamina. In addition, by blocking laminin at precise times in development, early events in embryogenesis that require laminin will proceed normally. We have identified laminin family members in the grasshopper that are similar to invertebrate and vertebrate homologs. Grasshopper laminin subunits have molecularly conserved functional domains and are predicted to have a similar tertiary structure compared to other laminins. Also, laminin is expressed during the outgrowth of central and peripheral pioneer neurons and is found evenly within the basal lamina. Laminin is expressed by hemocytes of mesodermal origin that migrate randomly throughout the lumen of the developing limb bud. In spite of the random migration of laminin expressing hemocytes, laminin deposition appears to be uniform in the basal lamina, which we speculate is held in place by interactions with the basal lamina and laminin receptors such as integrins. Integrin subunits are expressed on the epithelial cell layer that overlies the basal lamina. In addition, integrin is found on mesodermal cells and on T i l pioneer neuron cell bodies, axons and growth cones.  24  Antibody and peptide perturbation of the nidogen binding domain of laminin (but not a cell adhesion motif) results in defects in axon pathfinding. Specifically, T i l neurons cease growth. Prolonged culturing of T i l neurons in the presence of lamininnidogen blocking reagents does not rescue the cessation in growth. Furthermore, stalled T i l axons were localized to the site of an important steering decision within the trochanter as determined with Semaphorin l a and HRP immunofluorescence. Analysis of laminin and the secreted Semaphorin 2a expression reveals that the basal lamina has not suffered any obvious structural defects as a result of laminin-nidogen blocking. Thus, the defect observed in these embryos is specific to laminin nidogen interactions and not a secondary effect of basal lamina disruption.  25  CHAPTER 2: CLONING AND EXPRESSION OF GRASSHOPPER LAMININ We have chosen laminin as a candidate permissive cue in an attempt to understand the mechanisms that underlie growth of neurons on permissive substrates. Laminin mediated neuronal growth is well documented in vitro (see chapter 1), however, defining the role of laminin in vivo has been more difficult. The T i l pioneer neuron pathway in the developing grasshopper limb bud is a suitable model for laminin based neuronal growth. In order to analyze the function of laminin, laminin homologs in grasshopper were first cloned, and their expression pattern was determined. A close study of the molecular characteristics of laminin and the distribution of laminin protein in the basal lamina provides insight into the function of laminin and lays the necessary groundwork to proceed with functional studies.  Introduction The basal lamina is a molecularly heterogeneous structure composed of many molecules including laminin, type IV collagen, perlecan, and nidogen. Both laminin and type IV collagen can self assemble in vitro to form networks (Yurchenco and Furthmayr, 1984; Yurchenco et al., 1985; Cheng et al., 1997) which are essential for the function of the basal lamina. In addition to binding to perlecan, laminin and type IV collagen networks interact through binding of the intermediary protein nidogen. The molecular hierarchy of the basal lamina is highly ordered and arises in part by these molecular interactions between basal lamina molecules, as well as through linkage to cell-surface receptors such as integrins and dystroglycan (Henry and Campbell, 1998; Aumailley et 26  al., 2000; reviewed by Colognato and Yurchenco, 2000). At the developing neuromuscular junction, the distribution of numerous laminin isoforms are temporally and spatially regulated, indicating the importance of precise extracellular localization of laminin isoform (Patton et al., 1997). Laminin is a heterotrimeric protein that consists of three distinct subunits, a, (3, and y. As a major constituent of the basal lamina, laminin is conserved at the molecular level in vertebrates and invertebrates. Importantly, domain structure and functional motifs are conserved in invertebrate forms of laminin (Fessler et al., 1987; Montell and Goodman 1988; Montell and Goodman, 1989; Kusche-Gullberg et a l , 1992; Henchcliffe et al., 1993). Furthermore, invertebrate laminins are abundantly found in the basal lamina (Fessler et al., 1987; Montell and Goodman, 1989; Kusche-Gullberg et al., 1992). Laminin subunit genes are expressed very early during development and are involved in many developmental processes, including cell migrations and organogenesis. Genetic analysis of laminin function has implicated laminin in organ development (Yarnitzky and Volk, 1995; Miner and L i , 2000) neuromuscular junction formation (Noakes et al., 1995a; Sanes et al., 1998; Patton et al., 1998), muscle development (reviewed by Allamand and Campbell, 2000) and axon growth and guidance (Garcia-Alonso et al., 1996; Forrester and Garriga, 1997). In the nervous system, laminin is expressed along axon trajectories and serves as a substrate for axonal growth (Powell and Kleinman, 1997). In vitro, localized concentrations and gradients of laminin can reorient the growth of neurons. (Kuhn e t a l , 1995;Halfer, 1996; Kuhn et al., 1998). Furthermore, neurons change their adhesive properties when on a laminin substrate by regulating integrin receptors (Cohen et a l , 1986, 1987; Hall et al., 1987; de Curtis et al., 1991; de Curtis and  27  Reichardt, 1993; Condic and Letourneau 1997; Ivins et al., 1998, 2000). Thus, the response of neurons to laminin in an in vitro setting is complex and dynamic. Loss of laminin in genetic models can result in a disrupted basal lamina, thereby confounding the analysis of laminin function in such studies (Garcia Alonso et al., 1996; Miner and L i , 2000; Smyth et al., 1999). In addition, genetic loss of a single laminin subunit could result in either lack of secretion of the entire heterotrimer, as there is evidence that all three chains are required for secretion (Matsui et al., 1995), or increased expression of other subunits that may compensate for the missing subunit (Noakes et al., 1995b; Patton et al., 1997). Thus perturbing laminin function while maintaining the integrity of the basal lamina poses a challenge. In order to circumvent these problems, we have used the developing grasshopper embryo to examine the developmental expression and distribution of laminin during the earliest stages of peripheral nerve development. In addition, we have begun to develop various blocking reagents that will allow us to examine the function of laminin during embryonic development. In this study we identified grasshopper homologs of the (3 and / chains of laminin and describe the distribution of laminin in the grasshopper embryo, particularly in relation to the establishment of a sensory axon pathway in the developing limb bud. From 30-35% of embryonic development, the limb bud of the grasshopper is a hollow tube that is composed of epithelium and an underlying basal lamina, which separates the epithelium from the mesoderm. Both the mesoderm and the epithelium at this stage of development are relatively undifferentiated, however, muscle begins to differentiate (Ho et al., 1983) and limb segmentation proceeds. During this stage of development, neural precursors arise from the epithelium and extend axonal processes  28  into the CNS using the epithelium and the basal lamina as substrates for migration (Anderson and Tucker, 1988). The basal lamina is a thin extracellular sheet that exhibits variability in thickness and structure depending on location in the limb bud and stage of development (Anderson and Tucker, 1989). As the limb grows, the basal lamina changes in thickness, suggesting a dynamic role in the development of the limb, possibly aiding epithelial morphogenesis, muscle development and neuronal migration. Enzymatic removal of the basal lamina results in loss of axonal adhesion to the substratum, and perturbs epithelial morphogenesis, indicating an important role for an intact basal lamina (Condic and Bentley 1989a). To date, no molecular components of grasshopper basal lamina have been identified, nor has their mode of deposition into the basal lamina been described. In this study, we have described the cloning and expression of laminin during development of the nervous system. Laminin is distributed evenly in the epithelial basal lamina and is expressed by migrating hemocytes. Laminin has been shown to be expressed by wandering hemocytes in Drosophila, as well as by many other cell types that remain stationary, including ectoderm, mesoderm, glial cells and mesectodermal cells found in the CNS (Montel and Goodman, 1989; Fessler and Fessler, 1989; KuscheGullberg et a l , 1992; Kumagai et al., 2000). In vertebrates, laminin is deposited into the epithelial basal lamina by both epithelium and mesenchyme, and in some cases only by the mesenchyme (Ekblom et a l , 1998). In both vertebrates and Drosophila, laminin immunoreactivity found within the basal lamina is often found directly adjacent to the tissue that expresses it. In contrast, we have demonstrated the expression of laminin by migrating hemocytes. At the stages examined, the expression of laminin solely by  29  wandering hemocytes represents a novel mode of basal lamina deposition and suggests secondary factors that ensure the even distribution of laminin in the basal lamina.  Methods Cloning of Laminin p and /chains in Schistocerca gregaria For RT-PCR, embryos at 35-40% of development were removed from their egg cases in saline and R N A was extracted using Trizol (Gibco-BRL). To avoid amplification of genomic D N A in the subsequent PCR step, total R N A was treated with RNase-free DNase. For reverse transcription, the reaction contained 1-2 Lig of total R N A , 200 ng of random hexamer, 0.5 mg oligo dT, or 20 pmol of downstream primer. Degenerate primers specific for the EGF repeats of domain V were a generous gift of Dr. Mark Seeger (Ohio State University). Nucleotide sequence of these primers is degenerate for the amino acid sequence C K C N G H A S for the forward primer and GQCPCK(D/E) for the reverse primer. The nucleotide sequence is as follows: forward primer TG(C/T) A A ( A / G ) TG(C/T) AA(C/T) G G N CA(C/T) GC, reverse primer TC(C/T) TT (A/G)CA N G G (A/G)CA (C/T)TG N C C . The annealing temperature was varied to account for the degeneracy of the primers, beginning at 60°C for 2 cycles and dropping 2°C Celsius every other cycle, until a final annealing temperature was reached of 50° at which 20 cycles were conducted. Primers were removed from the PCR products prior to cloning using a Qiagen PCR Purification kit. Products were cloned into T-tailed pCR 2.1 (Invitrogen). Clones were digested with EcoRl and sequenced. Sequences were  30  entered into N C B I B L A S T x . Clones that showed homology to the laminin (3 chain were used as probes to screen c D N A libraries. A 600 bp clone of the grasshopper laminin (3 chain was used to screen an oligo dT primed lambda gt 11 library made from embryos at 40% of development (provided by Kai Zinn, California Institute of Technology) and an oligo dT primed lambda Z A P library made from nerve cord of embryos at 50% of development (provided by Michael Bastiani, University of Utah). Library screens were conducted as per Sambrook et al., (1989) In the case of clones from the lambda Z A P library, insert size of positive clones was analyzed by excision of the pBluescript plasmid and restriction analysis. With the lambda gt 11 library, inserts were amplified using sets of primers that either were specific to flanking lambda D N A or were specific for internal laminin (3 sequence, or a combination of the two. Larger clones were amplified using the Expand Long Template PCR System (Boehringer Mannheim). Positive clones were sequenced with an internal lambda primer and laminin specific primers derived from sequences obtained with the lambda primers. The Nucleic Acids Protein Services Unit at the University of British Columbia sequenced the majority of clones. Overlapping laminin P sequences were assembled in Assemblyline, translated with MacVector and aligned to known laminins with ClustalW.  31  Northern Analysis Total R N A from embryos at 40% of development (corresponding to a high expression of G-Laminin (3) was Trizol extracted (BRL). 27 ug of total R N A was electrophoresed in a 1% denaturing formaldehyde agarose gel and transferred to HybondN nylon membrane (Amersham). A single stranded DIG labeled D N A probe was generated by PCR using the laminin [3 PCR clone as a template.  In situ hybridization Linearized partial cDNAs for the (3 and 7 chains (domains III for both), were used for single stranded PCR in which a single primer was used with the Boeringer Mannheim DIG labeled PCR kit to generate DIG labeled probes. Both sense and antisense probes were generated in this fashion. Dot blots were used to determine incorporation of DIGlabeled nucleotide. Prior to use, probe was boiled for 40 to 60 minutes to generate smaller fragments. Embryos were dissected in saline and fixed for 50 minutes in P E M F A (0.1M PIPES [pH 6.95], 2.0 m M EGTA, 1.0 m M M g S 0 , 3.7% formaldehyde), and 4  washed 3X 1 minute, 3X 10 minutes in PBT ( I X PBS, 0.1% Triton X-100, 0.1% BSA). Embryos were incubated overnight at room temperature in hybridization buffer without probe (50 % formamide, 5X SSC, 100 u.g /ml Sonicated salmon sperm D N A , 50 pg /ml heparin, 0.1% Tween-20). Pre-hybridization buffer was replaced with fresh hybridization buffer containing 50 ng/ml boiled probe. Embryos were hybridized overnight at 72°C. Embryos were washed as follows: 5X SSC for 5 min, 0.2X SSC, 72°C, 0.2X SSC, room temperature, 5 min. Embryos were further washed for 5 minutes at room temperature in  32  buffer B l (0.1 M maleic acid, 0.15 M NaCI, pH 7.5), and buffer B2 (Buffer B l , 1% milk powder) for 60 minutes at room temperature. Embryos were incubated in antidigoxigenin antibody (Boeringer Mannheim) 1:5000 in buffer B2 for 1 hour at RT, followed by 2X 30 minute room temperature washes in buffer B l . Embryos were equilibrated for 5 minutes in buffer B3 (100 m M Tris-HCl pH 9.5, 100 m M NaCI, 5 m M MgCl ), and reacted in buffer B4 (buffer B3, 0.3375 mg/ml N B T , 0.175 mg/ml BCIP) 2  overnight at room temperature in the dark. Embryos were cleared in 70% glycerol and mounted in 100% glycerol.  Generation of Fusion Protein Constructs A 428 bp Eco R l / B s t Y l fragment of domain III of the (3 chain of grasshopper laminin was gel purified and subcloned into an EcoRl/BamHl cut p G E X 4T1 vector (Pharmacia Biotech). Similarly, a 561 bp Eco R l fragment of domain III of the / chain of laminin was subcloned into an EcoRl cut p G E X 2T. Cloning junctions of the constructs were sequenced with pGEX-specific primers to confirm orientation and frame. GST-fusion proteins were purified with glutathoine linked agarose beads (Sigma) as per Smith and Johnson, (1988). Protein was concentrated prior to injecting rabbits by dialyzing against PEG 8000 in 6-8 kD dialysis tubing. For initial injection, 0.5 mg of protein in 500 ml was emulsified with equal volume of Freund's complete adjuvant (Sigma). For all subsequent injections, 0.1 mg of protein in 500 ml was emulsified with equal volume of Freund's incomplete adjuvant (Sigma). Rabbits were housed and cared for at the University of British Columbia Animal Care Center. A l l bleeds were processed as per Harlow and Lane (1988). 33  Antibody Staining Embryos were dissected out of their egg cases in saline, and the amnion was removed, and staged as per Bentley et. al (1979). Embryos were fixed as with in situ hybridization. Embryos were blocked for 1 hour at 4°C in either PBT-5% normal goat serum, or PBT- 5% normal donkey serum, depending of the host of the secondary antibody. Primary antibodies, see below, were incubated overnight at 4°C, followed by several washes in PBT, and secondary antibody incubation at 1:250 in PBT for 1 hour at RT. Embryos were again washed in PBT and mounted in Slowfade antifade (Molecular Probes). Primary antibody concentrations were as follows: Goat and Rabbit anti-HRP 1:500, laminin 71:500, mes-3 1:4, 3H12 1:20,15 1:4, anti-Repo 1:250. Goat anti-HRP and Rabbit anti-HRP were from Jackson Immunoresearch and the monoclonal antibodies mes-3,15, and 3H12 were courtesy of Eldon Ball (Australian National University), and repo antibody was courtesy of Sarb Ner (University of British Columbia). Secondary antibodies used in this study were all purchased from Jackson Immunoresearch. They were: FITC conjugated donkey anti goat, FITC conjugated donkey anti rabbit, Cy3 conjugated goat anti mouse, FITC conjugated goat anti rabbit. For double labeling, primary antibodies were incubated together with embryos overnight at 4°C. Secondary antibodies were also incubated together for 1 hour at RT.  Pre-absorption of sera with fusion protein 10 pg purified fusion protein was incubated overnight at 4° Celsius with 1 pi crude sera in a final volume of 100 pi in PBS. The following day, embryos were stained as per protocol using the pre-absorbed sera. As controls, y sera was pre-absorbed with  34  the (3 fusion protein, a fusion protein that contained C. elegans alpha spectrin repeats (kindly provided by Ken Norman), and PBS. GST-reactive antibodies were removed from the sera by incubating with 1/20 volume GST-acetone powder overnight at 4°C. Supernatant was collected after centrifugation at 10,000xg for 10 minutes and filtered through a 0.4 mm filter.  Confocal Microscopy Confocal immunofluorescent images were collected on a Nikon Optiphot-2 microscope using the M R C 600 Confocal system (Bio-Rad) equipped with a Krypton/Argon laser.  The images collected from the confocal microscope were  captured in a 768X512 pixel field of view with the optical sections collected at 0.8 urn intervals. The confocal images were composed of a 100 to a 150 optical sections for each embryo. Data collected from the confocal microscope were analyzed in N I H Image 1.61 and Adobe Photoshop 5.0 was used for presentation. Confocal microscopy was conducted at the Biosciences Electron Microscopy Facility at the University of British Columbia.  Time Lapse Nomarski Microscopy Embryos from 33-36% of development were dissected from their egg cases in saline as described. Embryos were immobilized on glass coverslips coated in 5mg/ml poly-L-lysine and cultured in RPMI media. Embryos were imaged with Nomarski Optics on an inverted Nikon compound microscope, equipped with a Princeton Instruments MicroMax C C D camera (Kodak chip K A F 1400). Images were collected and analyzed using MetaView imaging software. Collected images (one every 3 minutes for 4 hours)  35  were compiled into a movie using MetaView software. The velocity of cells was determined by measuring the distance the cell traveled and dividing by the elapsed time. At the end of each time lapse period, embryos were fixed and processed for laminin immunofluorescence as described above.  Results Laminin family members in Schistocerca gregaria Using a combined approach of RT-PCR and cDNA library screening, grasshopper laminin sequences for the P and / chains of laminin were identified. Low stringency RTPCR with primers degenerate for domain V of the laminin (3 chain yielded portions of the P and y chains of the laminin heterotrimer. Additionally, eight overlapping cDNA clones of the P chain were isolated from cDNA library screens and sequenced. Canonical laminin P chains consist of a domain structure (similar to the a and y chains of laminin) of six domains numbered I though VI with an a domain between domain I and II. 3510 bp of laminin P sequence was cloned and sequenced which spanned domains I-IV and the a domain, but lacked domains V and VI.  Laminin P chains have been isolated from  many organisms and a shorter P chain in mouse, the P 3 chain does not contain domains V or VI. (Utani et al., 1995). However, no shorter P chains have been identified in Drosophila or C. elegans (Myers et al., 2000; Hutter et a l , 2000). Therefore, it is predicted that grasshopper laminin P indeed contains all 6 laminin domains, but that the 5' end evaded cloning due to technical constraints. However, the entire 3' end of the clone was identified, including the poly A tail, preceded by 3' UTR. In addition, the sequence within the PCR clone that was used to screen the library, and was used in  36  subsequent experiments, has no sequence errors and was confirmed by several c D N A clones as being part of domain III The predicted domain structure of grasshopper laminin (3 aligned to both mouse and Drosophila laminin (3 is shown in figure 4A. Once the 5' to 3' orientation was established, domain identity was based on homology to other laminin (3 chains. The most 5 prime end of the (3 sequence is homologous to domain IV of Drosophila and mouse P chain. Domains III, II, and I appear successively further along the 3' end of the sequence. Domain III of grasshopper laminin P begins at amino acid 179 and ends at amino acid 577. Domain II spans amino acids 578-795, the alpha domain spans amino acids 796-821, and domain I spans amino acids 822-1177. In the case of domain I (the most 3' end of the cDNA), the last amino acid that confers homology to the last amino acid on the Drosophila /3 chain is followed by a series of stop codons, 3' untranslated region, and the poly A tail. The region of grasshopper laminin P that was cloned consists of domains I-IV and the domain order and overall structure is similar to other laminins. Domains I-IV of grasshopper laminin P is 45% identical to domains I-IV of Drosophila laminin P and 37%i identical to mouse laminin P (figure 4A). Evident in figure 4A, the homology of grasshopper laminin diverges in domain I. Domain I is only 32% identical to domain I of Drosophila laminin whereas domains a to IV are 51% identical to domains a to IV of Drosophila laminin. Other laminin P sequences are also divergent in domain I compared to other regions (for example Drosophila to mouse in this region, see figure 4A). Therefore, this area does not appear to be very conserved throughout evolution. However, similar to Drosophila laminin P, tertiary structure analysis of  37  Figure 4: Alignment of grasshopper laminin deduced amino acid sequences with mouse and Drosophila laminin. (A) Partial grasshopper beta chain sequence (G-LN Beta) is aligned to mouse ( M - L N Beta) and Drosophila (D-LN Beta) sequences. Shaded areas indicate identity. Domain III, containing EGF repeats, was used to generate polyclonal antibodies (single bar). A box indicates the conserved cell adhesion site (YAGSR). (B) Partial grasshopper gamma chain sequence is aligned to mouse ( M - L N Gamma) and Drosophila (D-LN Gamma). The entire region, homologous to Domain III of the full length gamma chain, was used to generate polyclonal antibodies. A nidogen recognition sequence (box), D P N A V , is completely conserved in grasshopper.  38  A Q-LN Beta D-LN Beta M-LN Beta  i I i  AOLEF P I NN IQQGMD YD I V I R Y E P Q V P Q TWE YQVDV I R P Q P VO f l S Q P C A N A I PODOTKYVFLSPD S R S V P V S P P Y C L E S E L V F T Y Q D I PRSMPYDAV I BTQS T S R Q D W B N A F ITL VBPDOVOP EGQCGE L A AAT S S E T R I P F S L P D R S B Q V Y A L N E V C L E A A Y L E F F I D N I P Y S M E Y E I L IBYEPQL PDHWEKAY t T V Q B P O K I P A S S RCQN- -TYPQODNQVYS L S P Q - S R Y Y Y L P R P Y C F E K  Q-LN Beta 80 D-LN Beta m M-LN Seta 8i  T l K B i D Q - E T D VK P S5 AA S5 IILYDSI L Y D S I AALLII P P B H PPEESSII P PF F F FQQQQ - AANNLHHQEF N N L H R Q E F E R Y O Y V Y R H S T L Y I R A A FWMS Y I F H I Y I E B K B H - DVj0S P T A T I L YD S LTL I PR I DVTP I FQQ S VLAD I RKKDYgKYNCKS SLYOMNYKSDP • M N B T » « L E L P Q Y T A S Q S B l E l P Y T F i H v l M l T C K i L O B I V QQ S Q DG E Y TUS AWE T l O W R C L EN - • R S V V K T P M T O  Q-LN Beta i s D-LN Beta i » M-LN Beta tee  K Y H H S N R F L R SWYYH- - S P A K C D P T Q 5 Y S A I C S E L O O A C T CK P N V VQ R R C D B C A P Q T Y O F G P E Q C R A C D C N S 1 0 A L D N F C D V Q K O Q N L D N I L 8 Y F V H D G A S M C N C M P T Q S L S K V C E S N Q Q Y C Q C K P N Y V Q B Q C D Q C A P Q T Y G F G P E G C K A C D C N S I O S K D K Y C O L 1 V C R N I I F S I S A L I H Q T Q L A C E C O P O O S L S S V C D P N Q G Q C Q C B P N V V G R T C N B C A P G T F Q F G P N G C K P C D C H LQQSAS A F C D A I  G-LH Beta 236 D-LN Beta 239  R A N T Y G R E C D O C Q P Q F W N F P N C Q G C T C N G H A D Y C D P O T O A C T E C R D F T L Q H N C D R C I E Q Y Y G D P R I Q I D I P C R P C P C VP N T Y Q R E C N Q C Q P G Y W N F P E C R Y Q Q C N Q H A A T C O P I Q Q T C I D C Q O S T T Q T S C O S C L D a Y Y Q N P L F Q S E I G C R P C H C FQG I Y A R G C D R C L P G Y W G F P S C Q P C Q C N Q H A L D C D T Y T O E C L S C Q O Y T T G H N C E R C L A G Y Y Q D P I I G S Q D H C R P C P C  M-LN Beta 2«i  J  Q-LN Beta 3it D-LN Beta 322 M-LN Beta 3 a  PaTADSGHSYATRCALOTI. TODVVCECDVC PETYASQLAHADGCSLDTRNNNMLCHCQEG PDGPOSORGFAPSCYQOPVTLOLACVOOP6  Q-LN Beta «K D-LN Beta <KM M-LN Beta « t  HTEOFHCEICKANYYGDAI NOGCAECVCDI LG GTTQDHCELCKDQFFGDALOQNCGGCECDFLG  Q-LN Beta « o D-LN Beta m M-LN Beta «i  CDPIGSTAEQCNLFDQQCTCKPQFQGRQCNECQANHWQNPTYKCHACECDPDQS LTMQCHRENGTCICLEQIQGEKCNECABQ C D P I Q A I HEOCNS Y T O a C Q C K P Q F Q O B A C N Q C O A H Y W Q N P N F K C Q PCECDQFQAADFQCDRETQNCVCHEGfGGYKCNECABQ  P  JDVCADNYFGNPETPQOSCRPCNCSKNIOLAIPGNCDARSQFCLOCLF SEICADNFFGNPDN-GQTCSKCECSNNYDLYDTQNCDRQTGACLKCLY SDDCASQFPONPSOFQGSCQPCQCHHNIDTTDPEACOKDTGRCLKCLY  TNNTAGPCDBNTQQCPCLPNYlQQKCDECKPNHWK IASOTGC6PCG - TNNTIAHCDRF TGOCPCL PNVQGV RCOQCAENHWK IASGEQCESCN  HTEQDHCQLCQVQYYGDALRQDCRKCVCNYLQTVKEHCNGSDCHCDKATOOCSCLPNVIGONCDRCAPNTWQL  ASGTQCGPCN  C N A A H S F G P S C H E F T O O C Q C M P G F Q Q B T C S ECOELFWGDPDVECRACDCOPflai E T PQCDQS TQGCVCYfiSYEQP R C D K C T RQ  Q-LN Beta 303 Y L Q S A P Q C S P C Q E C F T N W D R t L N E L P . 0 Q T N R I ( Q D A 5 K t K Q T Q A T Q A Y T R E F D Q M D-LN Beta 363 YI Q Q F P H C S P C G E C F N N W D L t L 5 A L E D A T T A T 1 L R A K E I K Q V G A T Q A Y T 5 E F S E L D M-LN Beta 575 Y S O V F P D C T I H H Q M A L K A I I G E I T N R T H K F L E K H A L I I S B V I | P | R E T v B s Y  QMLSNTSISSQDLESLNKLY  RNLLONTSY3LYOIEKLDYET  D I L A G S P A A E P - L K N I G I L F  Q-LN Beta 516 D-LN Beta 648 M-LN Beta 6S7  I L t A H E A L R D U R Y K K D R t N A T A L G L K E N A T R L G E A N Y EGA L N- - - -L T R E AWE BSE GSLBDGU0ASHGRLSET6QNiD0IYNSLSLSGYELE5l.0NHSRLYGGL5KELKENG!OLOESNIEQALN - - - - LTBHAYEBVS lEAFKt TKDVT E K M A O V E V KLTBTASQS NS T A O l O A H A E A E S I O K I V H A I Q t E F I K N I D S O H B D S I T K Y F O M S L B A E K  Q-LN Beta 725 D-LN Beta 727 M-LN Beta 740  KAQQI A D D S G S A I A D S E R Q C K B A E T L Y N R T S N Q F L A L Q D E N T E A L A D L E T K L L E L E E Q I P D I N N Q Y C Q K ^ GOPCOKLCQG NLSTLKDEANELASNTDBNCKBYENLSNKIQAEADDLAN-NNKLI EDYRAELTSLTSQIPELNNQVCGKP- -QDPCD5LCGGR Y N A S T T D P H S T Y E Q S A L T R D B V 6 D L M L E R E S P P K E Q Q E E G A R L L D E L A Q K L Q S L D - - L S A A A Q M T C G T P P Q A D C S E S ECQGP  Q-LN Beta sto D-LN Beta soe M-LN Beta 821  NC R T D E G E K K C O O -  6DL.RGTLNHSSHG1.DDV6K1  LQNATGR  AQCGSCGQ - L S C E Q a A V T M A E B A l l F l E M R K•T lEEDD J l E — D U L S G Y S Q A RQ E T Y N K N T L l E A Y D»A L E A R U R S H D A G C G H C G G F L S C E H G A K T H S E E A L K V A K O A E T A 1T --•KDQAOQT IRALTQAKLNASEAYEKAKRGFEQSERYLNQTNA L AEY E Q LS KM V S E A K V R A D E A K G N A Q D V L LK T H A T K E K Y D K - P G C G G L V T V A H S A W G K A M O F D R D• LlA  LP  Q-LN Beta 882 D-LN Beta >S4 M-LN Beta 902  YTTRTQNFIDELQKFFDEEGASPSDtRT  Q-LN Beta 90s D-LN Beta « 7 M-LN Beta 983  DAAKAGA EG1 t E T A K L NBTIEKADKILDSlNS KRASKSATDYKYTADMVK  Q-LN Beta 1048 D-LN Beta 1050 M-LN Beta lots  F L Q N A H D A B l V A Q f E K L E A l V Q — A I QV - S C K T Q l D R A A A E VH K R A S RS N E A H E B A O S L U K K SWMMsB S L R K S K E L D O M K tMKMDRDAKEITKEAGSVKLEAMRARGEAN-NLQSATSATNQTLTDRASflSENABEBAKGLLGRASKLTVDTNAKLKDLNOLQ AAQN5GEA6Y(EKVVY$VKQNADDVKKTLOQELDEKYKKVESLIAQKTEESADARRKAELLQNEAKTLLAGANSKLGLLEOLE  Q-LN Beta 1130 D-LN Beta 1132 M-LN Beta 1151  K B V l . S K N I E t . e P g S I T Y L A Q N I N N T I A S L T D I f V l L M E T N D S l T K A K S l . E E Q A  NlKLAENLFIALNNFQENKTABPSESKEI S N E D L R H l l KQ I R N F L T B D S A D L D S I E A Y j  O K T t D L D L K L E P E E 1 E T L Q D Q I NRAVSSL KNVEA ) IYRTKPDLDRVNNIQS I A NEVIKSGNASTPQQLQNLTEDIRERVETISQVBVILQQSAADIARAELILEEA RKANNOISYAEROLTQlASGTEDAOGKANETVYEVNFLGARLKPLOTQ QQANSN1ELAQGDLEKIDEETTSAEAPANNTAOQVEKtAKKVQKLQNN IKHDEHOGTQNIHSIEKETAASEETLTMASIR  I SKIERNVEEBKBK  YDQHDKILROLNIBIDHlIDRMNKYQGYINI  RSEYYRSC HIM RQ S H V R Q C K Y A Y Y S TC IEDNQXYLEOKAQELYRIEGBYRSLIKDISI V L N K N O O L  L RLQA81QPLNKELNEHL  I  B Q-LN Gamma D-LN Gamma M-LN Gamma  CKCNGHAD I C O V E T G R C I C O H N T A E D N C D H C A R Q Y Y Q N A L O Q T A Y D C K P C P C P K O G P C IOL-QDETI ICLECPKQYGGSR CDCHGHAD t C D 3 E T G R C I C O H N T H G D N C D O C A K Q F Y Q N ALGOT P M D C K R C P C P N D G A C L O I - N E D T V I C T E C P K G Y F G B R C T C N G H S E T C O P E T G V C D C R D N T A G P H C E K C K D G Y Y G D S T L G T S S O C O P C P C P Q G S S C A I VPKTKEVVCTHCPTQTAQKP,  G-LN Gamma D-LN Gamma M-LN Gamma  CDLCSDGYFQDPTQKFQSVKL.COPCKRNDNV C E O C S D G F F Q D P T G L LGEVOTCKSCDCNGNV CELCDDGYFGOPLQSNQPVHLCRPCOCNDNll  Q LN Gamma D-LN Gamma M-LN Gamma  COCYPPGTVET3FGPPVODOISGOCPCK  C S C Y E A Q T E Q D E Q S I T RCDOVTGOCOCK CACNPYGTVOOO- - - S S C N P V T G O C O C  3NCNRTTQECLKCIYNTQGPECDSCIPGYFQDALA-LPKGDCKP 3NCNRTTGECLKCIHNT AG E H C D O C L S G H F G D P L A - L P H G R C D R SNCNRLTQECLKCtYNTAGFYCDRCKEGFFGNPLAPNPADKCKA  grasshopper (3 indicates a helical configuration in the last 600 amino acids, consistent with domains II and I forming a coiled coil (MacVector subsequence analysis). Domain III of grasshopper laminin (3 has 6 predicted EGF repeats, the same number predicted for domain III of Drosophila laminin (3. Furthermore, domain III of grasshopper laminin contains a partially conserved motif that is involved in cell adhesion, cell migrations and growth cone extension (Graf et al., 1987; Hopker et al., 1999). This amino acid sequence of this site, YIGSR, is conserved in grasshopper (YAGSR) (figure 4A). Northern analysis reveals a band at 8 kb, consistent with the large size of other laminin (3 chains (figure 5F). A second laminin chain was isolated from the RT-PCR screen. The deduced amino acid sequence of this clone shows a high degree of homology to domain III of the y chain of laminin (figure 4B). This clone is 67% and 55% identical to the Drosophila and mouse domain III of 7 laminin respectively. Antibodies raised against GST-fusion proteins and in situ hybridization results clearly demonstrate an overlap of (3 and / expression (this study) and (3 and / antibodies react with the laminin heterotrimer on a Western (figure 12D). A nidogen binding site on the 7 chain, D P N A V , is completely conserved in the grasshopper 7 chain of laminin (figure 4B).  Expression of laminin in the developing embryo Electron microscopic examination of the basal lamina has shown that it is already present by 30% of development, although its thickness throughout the limb bud is quite heterogeneous (Anderson and Tucker, 1989). However, it is unclear whether laminin is also distributed throughout the limb at this time or whether it exhibits any spatial heterogeneity. To answer this question, the expression pattern of laminin during  40  Figure 5: Expression of laminin in the developing grasshopper embryo. In situ hybridization (A-E) of laminin during development. (A). At 36% of embryonic development, laminin is expressed in mesodermal cells within the lumen of the developing limb bud (arrows) and in the pleuripodia, below the limb bud (arrowhead). In contrast, the epithelial layer (EL) is negative for laminin message. Distal is to the right, proximal is left, dorsal is up and ventral is down. (B). A control limb bud at 38% of development after hybridization with a sense probe. (C, D). Two high magnification examples of laminin positive mesodermal cells that have a distinctive morphology. Positive cells (arrows) exhibit filopodial like cellular processes (arrowheads). (E). Laminin message is expressed in mesodermal cells of the antennae (arrows) but not in the epithelium (EL). (F). Northern blot of grasshopper R N A from embryos at 40%) of embryonic development hybridized with a laminin antisense probe. A faint band at 8 kb (astericks) is indicated. Scale bars: (A, E), 30 urn, (C) 10 \xm. E L epithelial layer.  41  42-  limb development was examined using in situ hybridization and immunocytochemistry. Single stranded DIG labeled sense and antisense D N A probes were hybridized in embryos from 30%-46% of embryonic development. A l l the hybridization results presented here are from antisense probes specific for the (3 chain, however, similar results were found with the y chain. Between the stages of 30% to 46%, mesodermal cells that are scattered throughout the limb bud (figure 5A) consistently express laminin. Many of the cells that produce laminin message have a very distinct migratory morphology, possessing multiple filopodial and lamellipodial extensions (figure 5C,D). Laminin expression was also found in the mesoderm of the antennae (figure 5E), mandibles (data not shown), and pleuripodia (vestigial limbs, figure 5 A), but was not found in the epithelium in any of these structures (figure 5A, E). No staining was observed with sense probes (figure 5B). Although the results obtained with in situ hybridization reveal the localization of laminin message, they do not determine the localization of laminin protein in the basal lamina. To confirm results obtained with in situ hybridization and determine the protein localization of laminin in the basal lamina, polyclonal antibodies were raised against GST-fusion proteins of domains III of both the (3 and y chains of laminin (see Materials and Methods). From 30% to 42% of development, laminin positive cells are observed throughout the embryo. Similar to the in situ hybridization results, the cells are mesodermal in origin and display a migratory morphology. These cells closely resemble cells that were labeled with antisense probes in that their positioning in the limb and their morphology is  43  consistent with cellular labeling obtained with in situ hybridization probes (compare figure 6D with 5C, D). Antibodies generated against both the (3 and / chains exhibit identical staining patterns.  -  At 33% of development, single mesodermal cells scattered throughout the embryo are highly immunoreactive for laminin (figure 6D). In addition, the basal lamina is also positive. While the number of laminin positive cells is small at this stage, their number increases as development proceeds and the limb enlarges (compare figure 6D to 7A). Similar to the results obtained with in situ hybridization, the epithelium was never found to be labeled with the anti-laminin antibodies. Consistent with the in situ results, the mesodermal cells have a characteristic morphology, including a filopodia and lamellipodia (figure 6D). In addition to being expressed when the Ti 1 pioneer neurons are extending to the CNS from the periphery, laminin is also expressed in the CNS when the first pioneer neurons (progeny of the midline precursor (MP) cells) are establishing the CNS neuronal scaffold (figure 6A-C, Bate and Grunewald, 1981). This expression pattern of laminin during pioneer outgrowth both in the CNS and the PNS suggests a common role in axon outgrowth and guidance for all neurons. • Electron micrographs of basal lamina in the developing grasshopper limb bud demonstrate that the basal lamina has varying thickness depending on location in the limb and the stage of development, suggesting that basal lamina constituents may be unevenly distributed, perhaps in a gradient (Anderson and Tucker, 1989). Therefore, the distribution of laminin in the limb was examined during a period when a number of neurons in the limb are establishing projections, including the T i l neurons. During this  44  Figure 6: Immunofluorescence of laminin in the CNS and the limb bud. (A-C). Laminin expressing mesodermal cells are found throughout the CNS, neighboring developing CNS neurons. (A-B) embryonic CNS at 28% of development double labeled with antiHRP (green) and anti-laminin (red). (A) HRP immunofluorescence of M P neuronal cell bodies flanking the ventral midline with axons extending laterally (arrows). (B) At this stage, laminin is expressed by mesodermal cells in the CNS (arrows). (C) Overlay of laminin and HRP immunoreactivity. (D) Confocal image of laminin immunofluorescence in the developing limb bud at 33% of development. Laminin is expressed by mesodermal cells (arrow) that appear to be migrating, note filopodia (arrowhead). Laminin is not expressed by the epitheilial layer (EL) but is found in the basal lamina (BL). Scale bars: (A, D), 10 urn.  45  time, laminin appears evenly distributed throughout the limb upon examination with both confocal and conventional microscopy (figure 6D and 9A). Therefore, although the basal lamina as a whole varies in thickness, laminin expression appears to be uniform, suggesting that the heterogeneity observed in electron microscopy is due to differential localization of other basal lamina molecules. As laminin y sera stains the basal lamina as well as mesodermal cells, it is possible that the diffuse basal lamina staining is due to non-specific staining. To address this, laminin ysera was pre-absorbed with laminin 7 fusion protein and embryos were stained as before. Also, laminin y sera was pre-absorbed against the laminin p chain fusion protein, a fusion protein for C. elegans alpha spectrin repeats, GST-acetone powder, and PBS without fusion. Embryos that were stained with laminin ysera that was pre-absorbed with laminin y fusion protein did not stain the embryo. Conversely, laminin y sera that was pre-absorbed with either alpha spectrin fusion protein, laminin P chain fusion protein, GST acetone powder, or no fusion protein (PBS), stained the mesodermal cells and the basal lamina in embryos (data not shown).  Laminin is expressed by hemocytes To identify the cells secreting laminin, a number of cell markers were used that recognize different mesodermal lineages in the grasshopper embryo. Also, antibodies specific for repo, a Drosophila glial homeobox protein, that recognize glial cells in the developing grasshopper (Halter et al., 1995) were used to determine i f glial cells express laminin as has been reported in other organisms (Montel and Goodman, 1989; Luebke et al., 1995; Lentz et al. 1997). Figure 7A, B depicts the distribution of repo positive and  47  laminin positive cells in the limb bud and ventral nerve cord at approximately 29% of development. The cells that secrete laminin in the developing limb bud are not labeled by anti-repo antibodies, as no repo positive cells are present in the limb at this early stage. However, consistent with previous reports, repo positive glial cells are observed in the CNS (figure 7A, Halter et al., 1995). At later stages of development, repo positive cells are found in the limb and are associated with peripheral sensory axons (figure 7D, E). In addition, numerous CNS glial cells are associated with the longitudinal tracts (Halter et al., 1995). In contrast, laminin positive cells do not associate with the peripheral sensory axons, indicating that it is unlikely that glial cells are secreting laminin. Insect mesodermal cells encompass many different cell types, including the blood cell precursors, hemocytes (which differentiate into plasmatocytes and crystal cells) (Lebestky et al., 2000) muscle precursors, and muscle cells. In order to precisely determine the identity of the mesodermal cells that are secreting laminin, embryos were double labeled with mesodermal and laminin antibodies. Two antibodies that were used, 15 and mes-3, were reported to stain muscle pioneer cells (Chang et al., 1983; Kotrla and Goodman, 1984). A third antibody, 3H12, labels both hemocytes and the basal lamina (Ball et al., 1987). In figure 8 and figure 9, embryos were labeled with laminin and either mes-3,15, or 3H12. Immunofluorescent images of laminin and 15 in figure 8 A , B indicate that these antibodies label distinct subsets of mesodermal cells. Unlike the cells that secrete laminin, cells that label with 15 are not single cells with a random distribution within the limb. On the contrary, 15 positive cells appear in chains and are associated with muscle groups and tendons, as previously reported (Chang et al., 1983; Ho et al., 1983; Ball and Goodman, 1985).  48  Figure 7: Glial cells do not express laminin. (A) Embryo at 29% of development labeled with repo antisera, which labels glial cells (B) Laminin immunofluorescence of a different embryo at the same developmental stage as the embryo in (A). Repo labeled cells are confined to the CNS at this stage of development (A) and do not label with antilaminin antibodies (B). (C-D)Limb bud at 42% of development that is double labeled with anti-HRP (C) and repo antisera (D). Overlay in (E) At later stages, repo positive cells are associated with sensory axons in the distal limb. At approximately 42% of development the Ta3 neurons have extended toward the T i l pioneer pathway (Keshishian and Bentley, 1983). At later stages, repo immunoreactive cells are found in the limb periphery, however, they are associated with sensory nerves (arrowheads in E). Scale bars: B and E, 30 Lim.  49  A subset of mesodermal cells recognized by the mes-3 antibody also express laminin (figure 9A, B). The mes-3 antibody appears to label two subsets of cells with distinct morphology, single cells randomly distributed in the limb (figure 9B) and larger, more brightly labeled cells that are in a fixed position in all embryos examined (arrows in 9B). These larger mes-3 positive cells resemble muscle pioneers that also label with the 15 antibody as reported (Kotrla and Goodman, 1984) and do not express laminin (figure 9A, B). Unlike the 15 staining pattern, the single cells randomly arranged in the limb do express laminin (arrow heads in 9A, B). The mes-3 antibody appears to label two populations of cells: single migratory cells that also label with laminin antibodies and larger, more brightly labeled cells that appear stationary. Muscle development in the developing grasshopper limb occurs by a process in which mesodermal cells aggregate in the appropriate region and fuse to form syncitial masses (Ho et al., 1983; Ball and Goodman, 1985). Thus, the single cells that label with the mes-3 antibody and the laminin antibodies could be the undifferentiated counterparts to the more mature muscle pioneer cells that are stained with the mes-3 antibody but not the laminin antibody. The loss of laminin immunoreactivity in the muscle pioneer cells could reflect a developmental regulation of laminin expression. Down regulation of laminin expression in mature muscle is also seen in other systems where the expression of laminin subunits depends on the developmental stage of the muscle (Patton et al., 1997). In grasshopper, this down regulation of laminin, a possible permissive cue, may assist the targeting of motoneurons to muscle and promote synapse formation. It is also possible that mes-3 antibody labels muscle pioneers, as previously reported (Kotrla and Goodman, 1984) as  51  Figure 8: Laminin is expressed by a subset of mesodermal cells that are distinct from muscle precursors. (A) Limb bud at 38% of development labeled with laminin anti-sera, with arrows indicating laminin positive cells. (B) A different limb bud at the same stage as (A) labeled with monoclonal antibody 15, a muscle pioneer marker. Arrows indicate 15 positive muscle precursors, which appear syncitial, compared to laminin reactive cells. Scale bars: A , 30 Lim.  52  5£  Figure 9: Laminin is expressed by hemocytes. (A-B) Confocal image of embryonic limb bud at 40% of development double labeled with laminin (A) and mes-3 (B) antibodies. Arrowheads indicate a subset of mes-3 expressing cells that are also positive for laminin. A l l of the laminin expressing cells are also labeled with the mes-3 antibodies. However, a subset of mes-3 cells that appear morphologically similar to muscle pioneers do not express laminin (arrows in A and B). (C-D) Immunofluorescence microscopic image of embryonic limb bud at 31% of development double labeled with laminin (C) and 3H12 (D). Both the 3H12 antigen and laminin colocalize in hemocytes in the limb bud (arrowheads in C and D). The basal lamina is also immunoreactive for 3H12, but in an uneven pattern (arrows in D) (Ball et al. 1987). Scale bar 30 urn.  54  55  well as another class of mesodermal cells that secrete laminin. To clarify the identity of laminin and mes-3 positive cells, an antibody that recognizes hemocytes was used. Double labeling of embryos with laminin antibodies and the monoclonal antibody 3H12, which labels hemocytes and basal lamina, was conducted (Ball et al., 1987). As previously reported, the 3H12 antibody stains both the basal lamina and hemocytes. Embryos double labeled with laminin antibodies and 3H12 demonstrate that all laminin expressing cells are also 3H12 positive (figure 9 C, D). Based on prior characterization of these antibodies, some conflicting information has arisen with regard to the identity of laminin secreting mesoderm. To summarize, cellular laminin immunoreactivity colocalizes with a subset of mes-3 positive cells, a muscle pioneer marker, and 3H12, a hemocyte marker, but not with a second muscle pioneer marker, 15 (table 1). Muscle development in grasshopper was extensively studied using the 15 antibody and dye injections (Ball and Goodman, 1985; Ho et al., 1983). Therefore this antibody is very well characterized. However, the mes-3 antibody is less well characterized , and although it does label muscle pioneers as reported (Kotrla and Goodman, 1984), it is apparent that mes-3 also labels hemocytes. Our results agree with and expand on those found by Ball et al., (1987). Hemocytes, which are insect blood cells, secrete components of the basal lamina, and we have shown that hemocytes express a major constituent of the basal lamina, laminin. Expression of basal lamina constituents (including laminin) by hemocytes has been observed in other insect systems (Le Parco et a l , 1986; Mirre et al., 1988; Fessler and Fessler, 1989; Kusche-Gullberg et al., 1992; Fogerty et al., 1994; Gullberg et al., 1994; Yasothornsrikul et al., 1997; Kumagai et al.,  56  Table 1: Summary of immune-fluorescent results Antibody Cell type anti-repo 15 mes-3 3H12  Glia Muscle pioneers Muscle pioneers Hemocytes  Cellular co-localization with laminin antibodies None None Subset All  57  2000). Since the distribution of laminin and the 3H12 antigen differ, the 3H12 antibody probably recognizes a basal lamina molecule that is different from laminin.  Embryonic hemocytes migrate randomly in the developing grasshopper limb Descendents of the hemocyte lineage comprise cell types that are involved in humoral and cellular immunity in the adult. Plasmatocytes are analogous to macrophages and phagocytose invading microorganisms, while crystal cells are involved in humoral immunity (Mathey-Prevot and Perrimon, 1998). In the grasshopper embryo, before the generation of the hemolymph, many cells are migrating, for example, muscle pioneer cells (Ho et al., 1983; Ball and Goodman, 1985), neuronal cell bodies, and neuronal growth cones. Several lines of evidence suggest that the hemocytes that secrete laminin are migratory. Firstly, laminin expressing hemocytes are always randomly distributed throughout the limb and do not appear to have a fixed location. Secondly, the cells that express laminin message and protein display a morphology that is consistent with migratory cells, namely, they have multiple filopodia and lamellipodia (figures 5C, D and 6D). Thirdly, other classes of mesodermal cells are migratory, such as muscle pioneer cells (Ho et al., 1983; Ball and Goodman, 1985). Lastly, evidence from Drosophila has demonstrated that hemocytes are highly migratory, originating in the head mesoderm and circulating throughout the embryo (Tepass et al., 1994). In order to confirm that the laminin expressing cells are migratory hemocytes, time lapse videomicroscopy was employed to directly determine the nature of migrating cells in the developing limb bud. Grasshopper embryos at 35-40% of development were immobilized on poly-L-lysine glass coverslips, and bathed in a supplemented  58  grasshopper culture media. Images of the developing limb were collected every 3 minutes over a typical duration of 4 hours. Images were played back and migratory cells were tracked. Several observations of these cells were made. While some mesodermal cells appeared stationary, a subset of mesodermal cells (usually single cells) were migratory (figure 10). The rate of migration was highly variable while the direction of the migration appeared to be random (figure 1 ID). Many cells were observed to migrate to one location, only to reverse the direction of migration to reside in their original location at the end of the video period. Other cells migrated to a given location and then ceased movement for the duration of image collection. The average rate of migration of mesodermal cells was 0.35 Lim/min +/- 0.12 with a range of 0.14 Lim/min to 0.9 Lim/min (n=9) (table 2). Multiple filopodia were observed on the migrating cells, rapidly extending and retracting, presumably sampling the local environment. Furthermore, migration of a cell was typically preceded by the extension of a filopodia in the direction of the impending migratory event, similar to neuronal growth cones. These cytoplasmic extensions are similar in morphology to the extensions that are visible with antibody labeling and in situ hybridization with anti-laminin probes. To determine if these migratory cells observed with time lapse video microscopy were also expressing laminin, embryos were fixed and prepared for immunocytochemistry with laminin antibodies after the time lapse period. The anti-laminin labeling confirmed that the cells that migrated through the limb also expressed laminin (figure 10A, B , and 11C). From 34% to 44% of development, laminin postitive hemocytes were observed in the developing embryo. In addition,  59  Table 2: List of migrating cells that were characterized based on distance traveled Cell 1 2 3 4 5 6 7 8 9  Stage of embryogenesis 34% 34% 40% 40% 32% 44% 44% 40% 44%  Distance traveled (urn) 12.6 29 10.8 14.4 21.6 18.9 14.4 18 19.8  Velocity (Lim/min) 0.22 0.14 0.28 0.34 0.55 0.9 0.2 0.23 0.33  60  Figure 10: Migratory hemocytes express laminin. (A) High magnification of migrating cells in the developing limb bud. Arrow tracks the current position of the cell in each panel and asterisks serve as a reference point for the initial position of the cell. Between 144 and 168 minutes, the cell in panel (A) undergoes a cell division, indicated by a second arrow. Second to last panel shows an immunofluorescent image of the same cell stained with laminin anti-sera. In 57 minutes, this cell migrated 12.6 Lim with an average velocity of 0.22 Lim/min. The trajectory of the cell is shown schematically in the last panel (B). A second cell, located in the dorsal femur, is displayed as in panel (A). This cell traveled 29 Lim over 207 minutes with an average velocity of 0.14 Lim/minute. Laminin immunoreactivity and schematic of cell migration are shown in second to last and last panels, respectively. Dorsal is up, distal to the right, scale bar, 10 Lim.  61  Figure 11: Several migrating cells in the limb bud express laminin. (A) Grasshopper limb at 35% of development was analyzed using time-lapse Nomarski optics and immunofluorescence. At the onset of the time lapse experiment, mesodermal cells are clearly visible beneath the epithelium of the developing limb bud. Numbers 1-7 (located directly above the cell) indicate migratory hemocytes in their initial positions. (B) At the end of the time lapse period (216 minutes), all seven cells have migrated to new positions. (C) After the time lapse period, the limb was fixed and stained for laminin immunoreactivity. The seven cells are again indicated. Each migratory cell was also positive for laminin immunofluorescence. (D) Nomarski image at the beginning of the time lapse period with cells in their initial positions indicated with numbers, and the trajectory of the migratory events shown schematically. Scale bar 30 Lim.  63  64  migratory hemocytes were also observed at these stages, suggesting that deposition of laminin occurs by these cells which are continuously migrating. However, since cells were occasionally observed to cease movement during the time lapse period, we cannot rule out the possibility that laminin is secreted from these cells once migration is transiently ceased. Thus, in addition to phagocytosing apoptotic cells, hemocytes migrate throughout the embryo and secrete components of the basal lamina.  Discussion Laminins in grasshopper Using RT-PCR and library screening, laminin (3 and y chains were isolated from grasshopper embryonic RNA. The regions of the (3 and y chains of grasshopper laminin that were cloned show a high degree of homology to other laminin chains. Importantly, the laminin (3 chain shares the same domain order as other laminins. Two functionally important motifs in domain III of the (3 and y chains are conserved in grasshopper laminin. Within domain III of the (3 chain, a cell adhesion and migration motif was identified (Graf et al., 1987). Recent studies have shown that this motif is biologically active in converting the attractive properties of netrin, a bifunctional guidance cue, to repulsion (Hopker et al., 1999). This cell adhesion site, YIGSR, is partially conserved in grasshopper (YAGSR) suggesting an evolutionarily conserved role for this site. Polymeric sheets of laminin in the basal lamina physically interact with other basal lamina constituents through direct interactions or indirectly through intermediary binding partners such as nidogen (Timpl and Brown, 1996). On the / chain of laminin,  65  domain III contains a nidogen binding site, (Mayer et al., 1993a; Gerl et al., 1991) which consists of the peptide sequence, D P N A V (Poschl et al., 1996). The D P N A V sequence is completely conserved in Drosophila (Montell and Goodman, 1989), and Drosophila laminin binds to mammalian nidogen (Mayer et al., 1997). The region of domain III of grasshopper laminin y that was cloned contains this putative nidogen binding site and is completely identical to Drosophila and mouse. This suggests that grasshopper laminin yhas the same molecular characteristics as other laminins and likely plays an analogous role. Expression of laminin in the embryo is temporally correlated with neuronal pathfinding. In the limb bud, a uniform distribution of laminin is observed as early as 28% of development, before the first neuronal pathways are initiated, and the expression is maintained through later stages of development, when other pathways are established. Laminin expression is also correlated with pathfinding of the first neuron in the CNS, the M P neurons, suggesting that laminin can play a role in the pathfinding of both peripheral and central neurons. The even distribution of laminin in the basal lamina and the ubiquitous presence of laminin from 28-42% of development suggest that it may provide a permissive cue to extending growth cones. The pioneer T i l neuronal growth cones use the basal lamina as a substrate for migration, have intimate filopodial contacts with it and are adherent to it (Anderson and Tucker, 1988; Condic and Bentley, 1989b). The close association of T i l neurons to the basal lamina suggests that the basal lamina contains guidance information. We propose that laminin is at least one basal lamina molecule that is assisting the directed outgrowth of T i l neurons.  66  Embryonic hemocytes migrate randomly in the limb bud and secrete components of the basal lamina Insect hemocytes are mesodermal derivatives that give rise to blood cells such as plasmatocytes and crystal cells in Drosophila (reviewed by Mathey-Prevot and Perrimon, 1998). Plasmatocytes are analogous to macrophages and migrate within the hemolymph and phagocytose invading microorganisms in the adult. During Drosophila embryogenesis, hemocytes migrate from their origin, the head mesoderm, and phagocytose apoptotic cells (Tepass et al., 1994; Zhou et al., 1995). We observed hemocytes secreting laminin early in development in grasshopper, before dorsal closure. The morphology of hemocytes suggested that they were migratory, and we demonstrated that hemocytes migrate with varying speed and direction and secrete laminin into the basal lamina. As laminin is a substrate for various cell migrations, hemocytes may not only secrete laminin into the basal lamina, but may also use it as a substrate for movements. Since laminin is uniformly distributed in the basal lamina and hemocytes migrate without any apparent direction, it could be suggested that laminin is a permissive substrate for wandering hemocytes. Insect hemocytes have previously been reported to secrete other components of the basal lamina (Le Parco et al., 1986; Ball et al., 1987; Mirre et al., 1988; Fessler and Fessler, 1989; Kusche-Gullberg et al., 1992; Fogerty et al., 1994; Gullberg et al., 1994; Yasothornsrikul et al., 1997; Kumagai et al., 2000). However, this is the first example of migratory hemocytes as the sole producers of laminin and represents a novel means by which a basal lamina is established. In vertebrates, laminin in the basal lamina that separates the epithelial mesenchymal junction is produced by both epithelium and  67  mesenchyme, both of which directly border on the basal lamina (reviewed by Ekblom et al., 1998). Typically, the cells that secrete basal lamina constituents abut the basal lamina and could play a role in the extracellular polymerization and distribution of laminin (Colognato et al., 1999). However in C. elegans, the incorporation of type IV collagen into the basal lamina occurs in locations that are distant from the tissue that is expressing type IV collagen protein (Graham et a l , 1997). In this study, we demonstrate that laminin is expressed by cells that are only transiently associated with the basal lamina in a given location at a given moment, suggesting that the proper polymerization and localization of laminin is ensured by cells that do not express it.  Establishment of even laminin expression by migratory hemocytes The basal lamina staining observed with laminin antisera demonstrates that laminin is evenly distributed in the basal lamina. The basal lamina lies between the epithelium and the mesoderm in the developing limb bud. Although the epithelium completely and evenly surrounds the basal lamina, it is strikingly negative with both in situ hybridization and antibody labeling of laminin. The even distribution of laminin protein within the basal lamina therefore appears to be contributed by circulating hemocytes. Anderson and Tucker (1989) have demonstrated with electron microscopy the presence of a basal lamina as early as 30% of development in the grasshopper limb bud. The basal lamina is heterogeneous in composition both spatially and temporally, for example it is thicker dorsally in the femur and tarsus at 35% of development (Anderson and Tucker, 1989). However, this heterogeneity is not reflected by laminin staining at the light microscope level, since laminin appears to be evenly distributed in the basal  68  lamina. Therefore, the varying thickness of the basal lamina must be due to uneven incorporation of another basal lamina molecule. Indeed, the 3H12 antigen is distributed in the basal lamina in an uneven fashion and is concentrated in dorsal quadrants in the femur and tarsus consistent with the E M studies (Anderson and Tucker, 1989). Interestingly, this basal lamina constituent is also expressed by hemocytes, indicating that the distribution of basal lamina molecules is highly regulated. Furthermore, these molecules are retained in the basal lamina often not adjacent to the cells that express them. How do cells that migrate randomly within the limb ensure the even distribution of one basal lamina molecule, laminin, at the same time as ensuring the distal and dorsal accumulation of the 3H12 antigen? In the case of both laminin and 3H12, high cellular expression is found on the hemocytes, suggesting that these two molecules may be highly processed and retained in the cell. Alternatively, (but not mutually exclusive) hemocytes maintain a high expression level to ensure the promiscuous production of these basal lamina constituents, and another factor guarantees the appropriate distribution in the basal lamina, for example restricted expression of a binding partner. In the case of laminin, another basal lamina molecule may be expressed by the epithelium, which evenly overlies the basal lamina. The correct localization of this molecule may bind to and sequester the laminin in the appropriate location. Laminin has many binding partners in the basal lamina, including nidogen and perlecan (Timpl and Brown, 1996). Furthermore, it has also been demonstrated that laminin receptors can serve as nucleating sites for laminin polymerization (Colognato et al., 1999). Mutations in both integrins and dystroglycan result in disrupted basement membranes (Henry and Campbell, 1998; Aumailley et al.,  69  2000; reviewed by Colognato and Yurchenco, 2000) suggesting that these laminin receptors are essential for proper localization and polymerization of the laminin network. As the basal lamina lies between the mesoderm and the epithelium, a receptor on either cell layer would suffice. In support of this possibility, we have recently demonstrated that (31 integrin is expressed in the epithelium (Chapter 3). Therefore, laminin distribution within the basal lamina could be established in a two step process. First, high expression of laminin by circulating hemocytes provides abundant amounts of laminin in the basal lamina. Second, the distribution of laminin could be controlled by binding to integrins or another class of receptors, which are evenly expressed by the epithelium. In conclusion, we have demonstrated the presence of laminin subunit genes in the grasshopper that bear sequence homology at the molecular level to other laminins. Laminin expression is coincident with axon outgrowth and is evenly distributed in the basal lamina. Laminin is expressed by hemocytes, which migrate within the limb with no apparent course, yet somehow contribute to the even distribution of laminin in the basal lamina. We predict that the role of laminin expressing hemocytes is to produce high quantities of the laminin heterotrimer, which is most likely polymerized and assembled into a basal lamina by other tissues, such as the epithelium. This study suggests that basal lamina assembly and localization can occur by a mechanism that does not rely on the stationary position of laminin expressing cells, and most likely involves other tissue types.  70  CHAPTER 3: LAMININ IS REQUIRED FOR T i l AXON GUIDANCE The cloning and expression of laminin revealed that grasshopper laminin shows a high degree of homology to other laminins at the molecular level. In addition, laminin was found abundantly in the basal lamina during the time of Ti 1 pioneer neuron pathfinding. This is consistent with the known roles of laminin during nervous system development, namely as a substrate of neuronal growth. The even distribution of laminin indicates that it is a permissive cue for migrating axons. The following studies utilize the insight gained from the molecular characterization of laminin to elucidate the role of laminin as a permissive substrate of axonal growth.  Introduction A growing axon encounters a wide variety of cues as it establishes a projection to its target. These cues are processed and integrated by the growth cone resulting in directed outgrowth. Generally, guidance cues can be considered to be instructive or permissive. Instructive cues typically have a restricted expression pattern and guide neurons by conferring either attractive or inhibitory information on the growth cone. The behavior of a growth cone in response to a specific instructive cue can be modulated by several factors. Some of these factors include the presence of other guidance molecules (Chen et al., 1998; Giger et a l , 1998; Takahashi et a l , 1998; Winberg et a l , 1998), expression of different receptors on the growth cone (Hamelin et al., 1993; Bashaw et al., 1999; Hong et al., 1999; Takahashi et al., 1999; Tamagnone et al., 1999), the state of intracellular signaling cascades (Ming et al., 1997; Song et al., 1998), the presence of 71  basal lamina molecules (Hopker et al., 1999), and regulated cleavage of guidance molecules (Hattori et al., 2000; Galko and Tessier-Lavigne, 2000). Growth cones are often in contact with permissive cues while responding to instructive cues. However little is known about the roles of permissive cues in directing growth cones steering decisions, particularly when they are encountering an instructive cue. Recent work has indicated that repeated exposure to various cues can affect a neuron's response to future cues, (Shirasaki et al., 1998; Matise et al., 1999; Diefenbach et al., 2000). Thus permissive cues could act on extending growth cones to modulate their behavior in response to a particular instructive cue. Presently however, this modulation of instructive cues by prior exposure to permissive cues has not been demonstrated in vivo. There are a number of permissive cues in the developing embryo that would make excellent candidates as guidance information modulators. One of the most widely characterized structures that influences neurite outgrowth in vivo is the basal lamina. The basal lamina is a complex array of many different proteins, including laminin, type IV collagen, nidogen and perlecan. Upon secretion into the extracellular space, a given basal lamina molecule associates with itself, as well as other members of the basal lamina, and these interactions constitute the network of the basal lamina. The integrity of the basal lamina is very important for the many functions it performs including acting as a molecular sieve, providing a rigid support for adhesion (withstanding tension) and serving as a substrate for cell migration. A major component of the basal lamina that is also a significant stimulator of neurite outgrowth, is the large heterotrimer, laminin.  72  Laminin consists of three subunits, a, p\ and y. The three subunits are secreted as a trimer that can further self assemble to form polymeric sheets (Timpl and Brown, 1996) . Evidence suggests that lack of one laminin subunit is sufficient to prevent secretion of the entire trimer (Smyth et al., 1999). This loss of the entire laminin molecule, in turn, can result in the disruption of the basal lamina as evident from subunit knockout experiments in Drosophila and mouse (Garcia-Alonso et al., 1996; Smyth et al., 1999; Miner and L i , 2000). These structural disruptions of the basal lamina integrity may possibly perturb or disrupt its many functions. Thus a genetic analysis of laminin function during specific development events, such as cell migration and axon guidance, may be complicated by the secondary affects on the basal lamina structure. Due to these experimental constraints, the role of laminin in directing neurons during development in vivo has been a difficult question to address. Laminin has long been known to be a potent promoter of neurite outgrowth in vitro (reviewed by Powell and Kleinman, 1997). More recently it has been shown to also act as a directional cue for migrating axons in vitro (Kuhn et al., 1995; Halfter, 1996; Kuhn et al., 1998) and has been implicated in directing axons in vivo (Garcia-Alonso et al., 1996; Forrester and Garriga, 1997). One class of laminin receptor in neurons, the integrins, are critical in mediating laminin induced neurite outgrowth (reviewed by Powell and Kleinman, 1997). Integrin expression is highly dynamic and may be regulated by a number of factors including laminin availability (Condic and Letourneau, 1997) , laminin conformation (Calof et al., 1994; Ivins et a l , 1998) and the developmental age of the neuron (Hall et al., 1987; Cohen et al., 1986; Cohen et al., 1989; Ivins et a l , 1998; Ivins etal., 2000).  73  In vitro paradigms have often been used to examine the complex interaction between laminin and integrins, typically with one class of neurons plated on one isoform of laminin in the absence of a mature basal lamina. Essential to investigation of laminin in axon guidance and outgrowth would be an analysis of laminin function in vivo. In the developing embryo laminin is found within the context of the basal lamina and has numerous extracellular binding proteins that may affect its interaction with cell surface receptors. For example, laminin contains a specific binding site on the y chain that is recognized by the extracellular matrix molecule nidogen (Gerl et al., 1991; Mayer et al., 1993a; Poschl et al., 1996) and the L A R family of receptor tyrosine phosphatases binds to laminin-nidogen complexes (O'Grady et al., 1998). Furthermore, L A R s have been implicated as an important receptor for correct axon guidance (Krueger et al., 1996; Desai et al., 1997; Gershon et al., 1998; Wills et al., 1999; Bateman et al., 2000; Sun et al., 2000; Tisi et al., 2000). In addition, laminin only has one nidogen binding site (Mayer et al., 1993a) and blocking of the nidogen binding site on laminin effects the morphogenesis of epithelial tissues (Ekblom et al., 1994; Kadoya et al., 1997). In this study, the ability of laminin-nidogen interactions to guide neurons was examined. Grasshopper embryos were cultured in the presence of antibodies and peptides designed to interfere with nidogen binding to laminin. Both antibodies and peptides perturbed axon guidance of the Ti 1 neuronal pathway, suggesting that both acted as competitive inhibitors of nidogen binding. Ti 1 axons failed to complete the pathway, and ceased outgrowth at the site of a steering decision. Examination of the distribution of laminin and a secreted guidance cue (Semaphorin 2a) suggested that the effect of the peptides and antibodies on T i l pathfinding was not due to basal lamina disruptions. This  74  study demonstrates the importance of permissive cues and suggests that in vivo, permissive cues may modulate the response of the growth cone to instructive cues.  Methods Antibody Staining Embryos were processed for immunocytochemistry as described (Chapter 2). Primary antibody concentrations were as follows: Goat anti-HRP 1:500, rabbit anti-HRP, 1:500,  anti-laminin y chain 1:500, anti-semaphorin l a 1:1, anti-p integrin, 1:20, goat anti-  HRP and rabbit anti-HRP were from Jackson Immunoresearch and anti-(3 integrin was courtesy of Salvatore Carbonetto (McGill University). Secondary antibodies used in this study were purchased from Jackson Immunoresearch. They are: Texas red and FITC conjugated donkey anti goat, FITC conjugated donkey anti rabbit, FITC conjugated goat anti mouse, Cy3 conjugated goat anti mouse, FITC conjugated goat anti rabbit. For double labeling, primary antibodies were incubated together with embryos overnight at 4°C. Secondary antibodies were also incubated together for 1 hour at room temperature. For integrin immunofluorescence, embryos were immobilized on glass coverslips previously coated in 5 mg/ml poly-L-Lysine and filleted along the proximal-distal axis of the limb. Filleted limbs are rolled open to expose the ventral epithelium, containing the T i l neurons. Embryos were fixed and stained with anti- p i integrin antibodies.  Pre-absorption of sera with fusion protein 10  ug purified fusion protein was incubated overnight at 4°C with 1 JLII crude sera  in a final volume of 100 (il in PBS. The following day, embryos were stained as  75  described (Chapter 2) protocol using the pre-absorbed sera. As controls, y sera was preabsorbed with the beta fusion protein, a fusion protein that contained C. elegans alpha spectrin repeats (kindly provided by Ken Norman, University of British Columbia), and PBS. GST-reactive antibodies were removed from the sera by incubating with 1/20 volume GST-acetone powder overnight at 4°C. Supernatant was collected after centrifugation at 10,000xg for 10 minutes and filtered through a 0.4 um filter.  Confocal Microscopy Confocal immunofluorescent images were collected on a Nikon Optiphot-2 microscope using an M R C 600 Confocal system (Bio-Rad) equipped with a Krypton/Argon laser.  The images collected from the confocal microscope were  captured in a 768X512 pixel field of view with the optical sections collected at 0.8 mm intervals. The confocal images were composed of a 100 to a 150 optical sections for each embryo. Data collected from the confocal microscope were analyzed in NIH Image 1.61 and Adobe Photoshop 4.0 was used for presentation. Confocal microscopy was conducted at the Electron Microscopy facility at the University of British Columbia.  IgG purification IgG fraction of immune and preimmune sera was extracted using a Immunopure Protein A IgG purification kit manufactured by Pierce. Sera was loaded onto column and washed as per protocol. IgG fraction was eluted with 0.1M glycine pH 2 and neutralized with 100 u.1 of 1M Tris pH 7.5. Absorbance at 280 nm was taken and the concentration determined using the equation lOD=0.75 mg/ml protein.  76  Dialysis Prior to culturing, IgG purified pre-immune and immune sera were dialysed against sterile RPMI overnight at 4°C. Sera was placed in 6-8 kD dialysis tubing which was placed in 500 mis of sterile RPMI media. After overnight incubation, the media was refreshed once and dialysis continued for another 5 hours.  Western analysis of fusion proteins 50 ng of purified fusion protein was electrophoresed at 200 volts in a 7.5% SDS P A G E gel (4% stacking gel) and electro-transfered to Hybond E C L Nitrocellulose (Amersham). Transfer buffer consisted of 25 m M Tris, 192 m M glycine and 20% methanol. The blots were blocked in 5% milk powder, PBS, 0.1% Tween-20 overnight at 4°C. Primary antibody was used at a dilution of 1:10,000 for (3, y, antisera and P and ypre-immune sera in 1% milk powder, PBS, 0.1% Tween-20. Primary antibody incubations were conducted at room temperature for 2 hours, followed by washes in PBS, 0.1% Tween-20. Secondary antibodies (HRP conjugated goat anti rabbit) were diluted to 1:1500 in PBS, 0.1% Tween-20 and incubated for 1 hour at room temperature. Following washes in PBS, 0.1% Tween-20 and PBS, blots were reacted in E C L detection buffer (Amersham), and exposed to Kodak X - O M A T X-ray film.  Western analysis of embryonic lysate Approximately 50 grasshoppers at 35-40% of development were dissected from their egg cases in saline and resuspended in I X RIPA buffer (with 85 Lig/ml PMSF, 0.5 ug/ml aprotinin and 1 ug/ml leupeptin). Embryos were centrifuged at 14,000 rpm at 4°C  77  for 20 minutes, and supernatant was retained. Western analysis was carried out as with fusion protein with the following exceptions: embryonic lysates were electrophoresed on a 6% SDS P A G E gel (4% stacking gel) overnight at 20 volts at 4°C. Gel was electrotransferred to nitrocellulose in 50 m M Tris, 380 m M glycine, 0.1% SDS at 30 volts overnight at 4°C. The rest of the western was carried out as in the protocol for the fusion protein western.  Culturing Eggs were sterilized in 70% ethanol and rinsed twice in sterile grasshopper culture media (RPMI, 4 u M 20-hydroxyecdysone, 0.4 m M CaCl , 0.4 m M M g S 0 , 2 2  4  units/ml insulin, 100 units/ml penicillin, 100 pg /ml streptomycin, 10% heat inactivated FBS, 2 m M L-glutamine, 0.45 m M sodium pyruvate, 1 m M oxaloacetic acid, 0.45% Dglucose, 0.12 M sucrose, pH 6.9). Embryos were dissected as before (see antibody staining) in grasshopper culture media. Embryos were cultured for 22-24 hours in the presence of blocking peptides or antibodies. For longer cultures, embryos were prepared as described for culturing, but with the following modifications: After 24 hours in culture, media was removed and fresh media was added, with the appropriate blocking reagents.  Blocking peptides The D P N A V peptide was designed based on grasshopper laminin deduced amino acid sequences (Chapter 2). As a control, randomly scrambled peptides were used (PANDV for D P N A V ) . A l l peptides were generated at the Protein Services Unit at the  78  University of British Columbia and dissolved in sterile H 0 . D P N A V and P A N D V were 2  used at a final concentration of 0.2 mg/ml.  Blocking antibodies A l l antibodies, including pre-immune controls, were prepared as follows: sera was IgG purified, and dialysed against sterile RPMI. 7 antisera and pre-immune were used at 1 u M , as determined with an A280 spectrophotometric reading.  Assessment of Basal Lamina integrity in blocked embryos Embryos were cultured in the presence of D P N A V peptide for 24 hours as described. After the culture period, embryos were fixed and stained for HRP and Semaphorin 2a immunoreactivity and HRP and laminin 7 antisera immunoreactivity. Embryos were visualized with confocal microscopy as described in chapter 2.  Results To determine the potential role of laminin during the outgrowth of T i l pioneer neurons, a time course of laminin distribution with immunofluorescence was conducted. Laminin expression is evenly distributed during development of the T i l pathway, suggesting that T i l growth cones are in constant contact with laminin during their extensive migration to the CNS. In the developing limb bud the T i l pioneer sensory neuron projection establishes a projection to the CNS by 35% of embryonic development. The pathway is stereotyped and consists of a series of sequential steering decisions (Keshishian and Bentley, 1983; Bentley and O'Connor, 1992). At approximately 30% of embryonic development, the newly formed Ti 1 neurons delaminate from the epithelium  79  and extend axons proximally toward the central nervous system, (figure 12A). After the initial proximal extension of the Ti 1 axons, the Ti 1 growth cones contact a preaxonogenesis neuron, the Fel cell at 32% and continue proximal growth (figure 12B). Subsequently, at 33%> of development, the T i l axons contact a second pre-axonogenesis cell, the T r l cell (figure 12D). Upon contact with this cell, the T i l axons make an abrupt ventral turn (figure 12F). In the trochanter epithelium, before turning proximally and extending into the CNS, the T i l axons contact another intermediate target, the C x i neurons, (figure 12H and Keshishian and Bentley 1983). As the T i l growth cones migrate they make contact with several substrates including the laminin rich basal lamina (Anderson and Tucker 1989; chapter 2), epithelium and several intermediate neuronal targets (Keshishian and Bentley 1983, O'Connor, 1999). Later in development, the T i l pathway serves as a scaffold for later arising neurons. The specificity of antibodies raised against the y chain of laminin was tested on Western blots. Sera raised against EGF repeats of the y chain of laminin reacted with / fusion protein (figure 13C), but not with a fusion protein of EGF repeats from the P chain (figure 13C). On the other hand, the sera raised against the EGF repeats on the P chain, did mildly cross react with the / fusion protein (figure 13C). On grasshopper embryonic lysates both the / antisera and the sera raised against the EGF repeats of the p chain recognize the same high molecular weight band (figure 13D) that represents the laminin heterotrimer, which is possibly complexed within the laminin network. Antibodies generated against laminin chains were used to determine laminin distribution during T i l axon outgrowth, from 30% to 35% of embryonic development. At 30% of development, when T i l neurons have delaminated from the epithelium, an  80  even expression of laminin in the basal lamina is already established in the distal region of the limb bud (figure 13 A). In certain preparations, laminin appeared concentrated in the distal tip of the limb, for example in figure 12 A , 12G and 121. In addition, laminin distribution appeared to decrease in proximal limb segments (for example in figure 13A, 12A and 121). However, this could be an artifact of the staining, as anti-HRP background immunofluorescence is also diminished in these areas (see figure 12B and 12J). The distal accumulation of immunofluorescence and the proximal lack of immunofluorescence were not reproducible, and a non-uniform distribution was never observed along the pathway of T i l growth cone migrations (figure 12A-H). At 38% of development, later arising "follower" neurons begin to migrate towards the T i l pathway, which is used as a scaffold. At this time in development, laminin appears to be evenly expressed, (figure 12 I, J) suggesting a similar role of laminin in guidance of pioneer neurons as well as follower neurons. In addition to being found in the basal lamina, laminin protein (as well as messenger RNA) is also found in migrating hemocytes. These cells secrete laminin as they migrate throughout the lumen of the limb.  Localization  of laminin  receptors  on Til growth  cones  and  axons  Laminin has been shown to have many receptors, including several classes of integrins, the L A R family of receptor tyrosine phosphatases, and a-dystroglycan (Powell and Kleinman, 1997; O'Grady et al., 1998; Montanaro et al., 1999). Integrins have been found to mediate laminin dependent neurite outgrowth (Tomaselli et al., 1990; reviewed by Powell and Kleinman, 1997). To investigate potential receptors that may be mediating laminin's effects on migrating neurons, immunolocalization of integrin receptors was conducted. Two different polyclonal antibodies were used, one that was generated from  81  Figure 12: Time course of laminin expression in relation to the T i l pioneer neuron pathway. Immunofluorescence of laminin (left panels) and HRP (right panels). From 32% to 38%o of development, laminin is expressed evenly in the basal lamina during the time that the T i l pathway is established. Note in some preparations, laminin appeared to accumulate in the distal tip of the limb bud (C, G, I) Scale bar in A , 20 pm.  82  Figure 13: Laminin is expressed by hemocytes at 30% and 35% of embryonic development in the grasshopper limb bud. (A, B) Immunofluorescent images of grasshopper limb buds at 30% (A) and 35% (B) of development that are double labeled with laminin y antisera (green) and HRP (red). (A) T i l neurons emerge from the surrounding epithelium at 30% of development (arrow) in a basal lamina (BL) that is rich in laminin. Laminin expressing hemocytes are visible in the lumen of the limb (arrowhead). (B). Confocal image of a limb bud at 35% of embryonic development, the T i l pathway (red) has been established and arrows point out the T i l cell bodies, which have separated slightly in this limb. Laminin immunoreactivity in the basal lamina (BL) is even, and laminin expressing hemocytes are abundant. (C) Western analysis of laminin P and y GST fusion proteins with y and P antibodies. Laminin y antisera is specific for the y fusion protein but does not recognize the P chain fusion protein. However, slight cross reactivity was observed with the P antisera and the y fusion protein (asterick) (D) Western analysis of lysate from embryos at 40% of development with P and y antibodies and pre-immune sera (PI). Both P and y antisera recognize a high molecular weight complex (polymerized laminin) in the stacking gel. The P and y antibodies recognize a similarly sized band. Pre-immune sera (PI) reacts with no bands. Scale bar in A , 10 urn.  84  <S5  the entire (31 subunit of integrin and another that recognizes the intracellular domain of (31 integrin. As controls, immunocytochemistry was performed with secondary antibody alone, which did not stain the embryo. On whole embryos, both antibodies stained epithelial cell boundaries, and since this staining was quite intense, T i l neurons could not be visualized. Therefore, limbs were filleted open and immobilized on poly-L-lysine coated cover slips and stained with polyclonal antibodies to the entire p i integrin subunit (figure 14B) and polyclonal antibodies to the intracellular domain of (31 integrin (figure 14C, D). In this preparation, p i integrin was found on T i l cell bodies, axons and growth cones (figure 14B, C). In addition, in the limb fillet preparation, integrin was expressed at epithelial cell boundaries, as well as on mesodermal cells (figure 14C, D). Expression of p i integrin on T i l growth cones suggests that integrins could be transducing laminin signals in the basal lamina.  Blocking a conserved nidogen recognition sequence on laminin disrupts axon guidance Laminin is found along the entire course of T i l axon trajectory (figure 13), suggesting that laminin is conferring information to the Ti 1 growth cones throughout their migration. Confocal analysis of laminin immunofluorescence in the developing limb bud basal lamina demonstrates that the distribution of laminin is even (figure 12B), suggesting that laminin may play a permissive role in axon guidance. EGF-repeat containing domain III of grasshopper laminin y contains a completely conserved nidogen  86  binding site (Gerl et a l , 1991; Mayer et al., 1993a; Poschl et al., 1996; Chapter 2). This site has been implicated in epithelial morphogenesis (Ekblom et al., 1994; Kadoya et al., 1997), and nidogen has recently been shown to direct migrating neurons (Kim and Wadsworth, 2000).  Previous studies have demonstrated that both antibodies and  peptides directed at this site interfere with nidogen binding to laminin (Mayer et al., 1993a). With this in mind, antibodies and peptides were designed to target this site, in order to disrupt laminin-nidogen interactions and to determine their role in axon guidance. Grasshopper embryos were cultured in the presence of these reagents at 30% of embryonic development. This allowed the embryo to develop unperturbed prior to the culture period, in order to prevent any secondary effects of disrupting laminin function. Therefore, it was possible to determine the role of laminin-nidogen interactions in growth cone guidance. Embryos at 30% of development were cultured for 24 hours at 30° Celsius in the presence of 1 u M IgG purified laminin y antisera and the synthetic peptide D P N A V (0.2 mg/ml). As controls, IgG purified pre-immune sera and the randomly scrambled peptide P A N D V at the same concentrations as y antisera and D P N A V , respectively were used. T i l neurons exhibited three types of pathfinding errors. However, only one error was significantly different from control cultures. This error, stalled axons, is characterized by the failure of both T i l neurons to initiate the ventral turn at the trochanter segment and to extend to the CNS (figure 15B and 16B). One or both of the T i l axons extending in the dorsal quadrant of the limb compartment typify the second type of abnormality, a dorsal projection (figure 15C). Finally, the third abnormality is characterized by a distal projection of one or both T i l axons from the cell bodies instead of extending proximally  87  (figure 15D). Importantly, for both blocking reagents, similar abnormalities were observed, suggesting that both reagents were acting by the same mechanism, probably as competitive inhibitors. The rate of disrupted T i l pathways when cultured with 1 LIM 7antibody was significantly higher than controls, at rates of 46 ± 3.7% (n=387, 6 independent trials, figure 17B) compared to 11.1 ± 2.2% in RPMI and 20.6 ± 4.5% with pre-immune sera; p= 5.2X10" for RPMI vs. y antibody, p= 0.0007 for pre-immune vs. y antibody (unpaired 6  student's t-test). Similarly, 41 ± 5.2 % of embryos that were cultured in the presence of 0.2 mg/ml D P N A V (n=337, 6 independent trials, figure 17B) exhibited disrupted T i l pathways, compared to 14.8 ± 1.1% for RPMI and 21 ± 2.8% for control peptide. P= 0.00031 for RPMI vs D P N A V and p=0.004 for control peptide vs. D P N A V . The most frequent error in the Ti 1 pathway after laminin-nidogen block was stalled axons, (compare figure 16A and 16B). The integrity of the growth cones and the T i l cell bodies suggested that cell death was not a factor in these experiments (figure 16B). In antibody and peptide blocked embryos, 89 ± 6.4 % (n=181) and 80 ± 6.2% (n=131) of total errors were of the stalled variety (figure 17D). The other errors were dorsal projections (at rates of 9.4 ± 6.7 % and 16 ±5.9 % for antibody and peptide respectively) and distal projections (at rates of 1.1 ± 0.67 % and 1.5 ± 1.3 % for antibody and peptide, respectively). However, a small number of dorsal errors were also found in control embryos. A comparison of the dorsal error rate in control cultures compared to experimental cultures revealed that there was no statistical difference between control and antibody treated (p=0.7) and control and peptide treated (p=0.5).  88  Figure 14: (31 integrin is expressed in the developing limb bud. (A-B) T i l neurons at 32% of development are labeled with anti-HRP antibodies (A) and p i integrin antibodies (B). Arrow indicates growth cone that is depicted in higher magnification in (A'). (B) (31 integrin immunofluorescence of the same neuron in (A). [31 integrin localizes to the cell bodies, axons and growth cones of the T i l neurons. (31 integrin is also expressed by epithelium and mesoderm, which accounts for the out of focus staining in this panel. Arrow in (B) indicates the growth cone that is magnified in (B'). (B') p i integrin is expressed on the growth cones of T i l sensory axons. (C) p i integrin staining is confirmed with a different antibody. In an embryo at 34% of development, p i integrin is expressed in T i l cell bodies (asterisks) and axons. Growth cones are out of the field. T i l axons migrate along an epithelium that is evenly expressing p i integrin at cell junctions. (D) Mesodermal cells, possibly hemocytes, are highly expressing p i integrin. Same embryo as depicted in (C), but at a slightly different focal plane to emphasize mesodermal staining. T i l cell bodies (out of focus) are indicated with asterisks. Scale bar in A , C, 10 urn.  89  A salient feature of the stalled axons is the location within the T i l pathway in which T i l axons stall. Stalled T i l axons were never observed proximal to the ventral turn, for example, never after committing to the ventral turn, or in the vicinity of the C x i cells. Therefore, it appeared that T i l axons either stalled before the ventral turn, or completed the pathway. Several mechanisms could account for the cessation of growth and the inability of T i l axons to complete their trajectory. One explanation is the possibility that these neurons are growing slower than in control cultures, and the culture period is terminated prematurely, preventing the axons from completing the trajectory to the CNS.  TM growth cones remain stalled after prolonged culturing We have established that blocking a nidogen recognition sequence on laminin results in stalled T i l axons. Growth rate of neurons in vitro is dependent upon substrate, for example, chick D R G neurons extend two times faster on laminin than on fibronectin (Kuhn et al., 1995). T i l growth cones intimately associate with the basal lamina in the developing limb bud, by filopodial sampling (Anderson and Tucker, 1988). Therefore it is possible that T i l growth cones exhibit substantially retarded growth as a result of the disruption of laminin-nidogen interactions. This would also account for the consistency of the location of the stall, since embryos are cultured for 22-24 hours. To address whether T i l axons were still growing but required a longer culture period in order to complete the pathway, embryos were cultured in the presence of D P N A V blocking peptide for 48 hours. When cultured for 48 hours, a similar number of aberrant projections were observed; 36 ±0.75% of axons were abnormal (n=141, p=0.0021 for  91  RPMI vs. D P N A V peptide, and p= 3.6X10" for control peptide vs. D P N A V , figure 17C). 5  The distribution of errors was not significantly different as 76.5±11.4% stalled, 21.6 ±11.7% extended aberrantly dorsally, and 2 ±1.4% grew distally (n=51, figure 17D). Therefore, despite the extended culture period, the T i l growth ones ceased growth at the same location within the limb at the trochanter limb segment epithelium.  Ti1 axons stall within filopodial range of guidance cues found within the trochanter The trochanter segment contains pertinent guidance information that acts in part to mediate the ventral turn, for example members of the semaphorin family (Kolodkin et al., 1992; Isbister et al., 1999). If T i l axons stall before the trochanter epithelium, then they may not be within filopodial range of important guidance information. If they stall within the trochanter, then they have access to semaphorins and other guidance information found there.  In order to firmly establish the location of stalled axons, two  criteria were used. First, Semaphorin l a is a transmembrane protein expressed in a band of epithelium in the trochanter segment at 33-35% of development (Kolodkin et al., 1992), and therefore was used as a marker for the trochanter epithelium. Second, the location of stalled T i l axons in relation to a pair of pre-axonogenesis neurons, the C x i cells, was used. These cells lie just proximal to the trochanter limb segment in the distal compartment of the coxa segment (figure 18A and figure 19A). The neuronal marker used in all blocking experiments, HRP, also labels these pre-axonogenesis neurons. Embryos were cultured in the presence of D P N A V blocking peptide and cultured overnight, fixed and stained for both HRP and Semaphorin l a immunoreactivity. T i l  92  axons were scored in relation to Semaphorin l a expressing epithelium (the trochanter, figure 18 and figure 19B) or in relation to the C x i cells (figure 18A and 19A). Using these criteria, 73% of stalled T i l growth cones resided within the trochanter, whereas 17% and 8.7% stalled before and after the trochanter respectively (n=23, figure 19D). Interestingly, axons that were found after the trochanter had not initiated the ventral turn, instead they had continued extending proximally (see figure 18C). These results would suggest that the majority of stalled T i l growth cones (81.7%, those that stalled in the trochanter and those that stalled past the trochanter) had access to guidance information within the trochanter, such as semaphorins, but were unable to respond to it.  Basal lamina integrity The complex arrangement of the basal lamina results from interactions of many different basal lamina constituents. Basal lamina molecules such as laminin and type IV collagen can self-assemble, in addition, other basal lamina components can bind to laminin and type IV collagen networks (Yurchenco and Furthmayr, 1984; Yurchenco et al., 1985; Timpl and Brown, 1996; Cheng et al., 1997). Genetic loss of basal lamina molecules results in disrupted basal lamina, for example in the case of laminin, and type IV collagen, and perlecan, but not in the case of nidogen (Garcia Alonso et al., 1996; Costell et al., 1999; K i m and Wadsworth, 2000; Norman and Moerman, 2000; Miner and L i , 2000; Murshed et al., 2000). However, antibodies to the nidogen binding site on laminin result in basal lamina disruptions in vitro (Kadoya et al., 1997). Since nidogen binds to both type IV collagen and laminin, it is proposed to serve as a linker molecule for these two basal lamina networks. Therefore, interference of laminin-nidogen  93  Figure 15: Schematic illustration of types of T i l pathfinding errors observed in cultured embryos. (A). The normal T i l pathway. (B). Abnormalities classified as a stall are characterized by both axons ceasing proximal growth. (C). In dorsal abnormalities, one or both T i l axons migrate into the dorsal quadrant of the limb and remain there. (D). In the case of distal abnormalities, one or both T i l axons exend distally to the epithelium in the distal tip of the limb bud.  94  Figure 16: Disrupting a conserved nidogen recognition sequence results in aberrant neuronal pathfinding. (A). Culturing in pre-immune sera or randomly scrambled peptides (see text) has minimal effect on Ti 1 pathfinding. Control culture demonstrating the T i l pathway as visualized with HRP immunofluorescence. (B). T i l axons stall when embryos are cultured in the presence of 0.2 mg/ml D P N A V peptide. T i l neurons have brightly labeled cell bodies, axons, and extended growth cones, indicative of healthy neurons. (C). Antibodies have access to the lumen of the limb bud during the culture period. Embryo after culturing with laminin y antisera with the addition of anti-rabbit secondary antibody demonstrates that the antisera has penetrated the limb bud and is localized binding to laminin epitopes found in the basal lamina and on hemocytes. Scale bar: (A-C) 20 pm.  96  Figure 17: Summary of laminin antibody and peptide blocking experiments on the y chain. (A). Illustration of the laminin heterotrimer indicating the site that antibodies and peptides were designed to disrupt. (B) Antibodies and peptides to the nidogen binding site on laminin disrupts neuronal pathfinding. In 24 hour cultures, both antibodies to the nidogen binding site and peptides result in and statistically significant increase in total T i l pathfinding errors (p=5.2X10" for y antibody vs. RPMI cultures, p=0.0007 for pre6  immune vs. y antibody cultures, p=0.00031 for D P N A V peptide cultures vs. RPMI cultures, and p=0.004 for control peptide vs. D P N A V ) . (C). After 48 hours in culture, T i l axons do not recover, and still exhibit pathfinding errors, similar to 24 hour cultures (p=0.0021 for RPMI vs D P N A V peptide cultures, and p=3.6X10" for control peptide vs. 5  D P N A V . (D). Total T i l pathfinding error was normalized to 100% and the total errors were broken down into categories. In both 24 and 48 hour cultures, the majority of errors were stalled axons.  98  interactions with antibodies and peptides, could result in a disruptions of the basal lamina. Aside from resulting in a disrupted basal lamina scaffold for migrating cells, basal lamina disruptions could alter the localization of secreted guidance molecules, for example members of the semaphorin family. To assess whether neurons ceased growth due to an alteration of the basal lamina, laminin distribution was examined in embryos cultured in the presence of blocking peptide. If the basal lamina had suffered a structural defect, then laminin immunoreactivity may appear disrupted. In laminin-nidogen peptide blocked embryos, immunofluorescence of laminin was similar to control embryos (figure 20A, B). Laminin protein was evenly distributed similar to control cultures indicating that the laminin network was not severely disrupted. Figure 20B depicts stalled Ti 1 growth cones in the trochanter, surrounded by a normal laminin distribution. Semaphorin 2a is a member of the semaphorin family of guidance molecules (Isbister et al., 1999; Bonner and O'Connor, 2000). Semaphorin 2a is secreted and is normally localized in a dorsal to ventral and distal to proximal gradient (figure 20C, Isbister et al., 1999). To determine if secreted guidance cue localization was disrupted in laminin-nidogen blocked embryos, peptide blocked embryos were labeled with Semaphorin 2a antibodies. In peptide blocked embryos, Semaphorin 2a distribution is similar to control cultures, suggesting that at least in the case of Semaphorin 2a, secreted guidance cue localization was intact in laminin-nidogen blocked embryos. Stalled T i l axons therefore have access to appropriately localized Semaphorin 2a (figure 20B, arrow), which repels T i l axons from the dorsal limb compartment.  100  Figure 18: Schematic depicting the three possible locations of T i l stalled axons when laminin is blocked. Stalled axons were scored in relation to Semaphorin l a immunofluorescence (gray band) which is coincident with the trochanter segment (Tr), or in relation to the C x i cells which are proximal to the trochanter in the coxa segment. (A). T i l axons could be found stalled femur limb segment, before the trochanter. (B). If T i l axons were found within the band of Semaphrorin l a expression, or just distal to the C x i cells, there were scored as residing within the trochanter. (D). If stalled axons had extended past the Tr, they were scored as such. Note that these growth cones did not turn, but extended abnormally proximally. Tr is trochanter.  101  Before the Trocanter  A Within the Trocanter  B After the Trochanter (but not turning)  joz.  Figure 19. Ti axons stall in a discrete location in the limb bud when laminin is blocked. (A-C) Laminin-nidogen blocked embryo that has been double labeled with HRP to visualize neurons, (A) and Semaphorin l a (B). Overlay in (C). In (C), T i l axons have stalled within the Semaphorin l a expressing epithelium, which is coincident with the trochanter (Tr) limb segment. Semaphorin l a expressing epithelium and the location of the C x i neurons were used to determine the location of 23 stalled T i l growth cones. (D) 73% of stalled T i l axons resided within the trochanter, site of the ventral turn. Scale bar in A , 30 Lim.  103  Tr  B Location of Stalled Til growth cones  Figure 20: Basal lamina and secreted guidance cue localization are intact in lamininnidogen blocked embryos. (A) Immunofluorescence microscopy of a control embryo that has been labeled with laminin antisera. (B) A D P N A V blocked embryo that has been double labeled with laminin (red) and HRP (green). T i l axons stall in the trochanter (arrow) and no structural defects in the basal lamina are observed . (C) Sema 2a, a secreted guidance cue, is distributed in a dorsal to ventral and distal to proximal gradient (Isbister et al 1999) in control cultures. (D) Sema 2a distribution is preserved in D P N A V blocked embryos. In this embryo, T i l axons (green) have stalled in the trochanter (arrow) and Sema 2a (red) is expressed in a dorsal to ventral and distal to proximal gradient. Scale bar in A, 20 pm.  105  Blocking Beta chain function has no effect on Ti1 pathfinding The bifunctionality of guidance cues, that is cues that are both attractive and repulsive, can be modulated by various factors, including levels of cyclic nucleotides, and receptor types on the growth cone (Hamelin et al., 1993; Ming et al., 1997; Song et al., 1998; Bashaw et al., 1999; Hong et al., 1999; Takahashi et al., 1999; Tamagnone et al., 1999). It was recently demonstrated that a cell adhesion motif on the laminin beta chain can convert netrin, a bifunctional guidance molecule from an attractive to a repulsive cue (Hopker et al., 1999). The amino acid sequence of this site, YIGSR, is partially conserved in grasshopper and is Y A G S R (Chapter 2). To address whether or not this site has a role in T i l pathfinding, we used polyclonal antibodies and peptides bearing the sequence Y A G S R as blocking reagents. Using the same methodology as described for the laminin-nidogen site, neither the beta antibody, nor the Y A G S R peptide had any effect on T i l pathfinding (figure 21). Antibodies were used at concentration ranges of 1 LIM to 3 u,M (figure 21 B and figure 22A) and peptides were used at concentration ranges of 0.2 mg/ml to 0.8 mg/ml without any appreciable effect (figure 2IB and 22B). Although both the (3 chain antibodies and the peptides had no effect on pathfinding, the pre-immune sera had a small effect on T i l pathfinding at 2 LIM (figure 2IB), which increased with increasing concentrations (figure 22A). This indicates the presence of a factor in the pre-immune that effects T i l pathfinding, that was not active in the immune sera. Due to the effect of the pre-immune at 3 u M , this control was not done at the 5 u M concentration (figure 22A), These results indicate that while this site is active in vitro, and alters netrin function, it is not important 107  Figure 21: Summary of laminin antibody and peptide blocking experiments on the (3 chain. (A). Illustration of the laminin heterotrimer indicating the site that antibodies and peptides were designed to disrupt. (B) Antibodies at 2 o M and peptides at 0.4 ng/ml had no effect on Ti 1 pathfinding.  108  Figure 22: Summary of results of different doses of antibodies and peptides designed to disrupt laminin P chain function. (A). At 1 p M , and 3 o M , P antibodies did not effect T i l pathfinding, although pre-immune sera at 3 LIM did effect T i l pathfinding. As a result, pre-immune was not used at the 5 LIM concentration, but the pathfinding error with the immune was not significantly higher than that found with 3 u,M pre-immune. Data for the 3 p M concentration was obtained from the average of two trials. (B) At 0.2, 0.6, and 0.8 mg/ml, Y A G S R peptides did not alter the course of T i l axons significantly. Data for all peptide concentrations were the result of averaging two independent trials.  110  Beta Chain (antibody block)  Beta Chain (peptide block)  in the formation of the T i l pathway. Presently it is not known whether netrins are found in grasshopper and if so, where and when they are expressed. Nonetheless, these results substantiate the validity of the laminin-nidogen blocking results in that simply by blocking laminin, regardless of the site, does not alter T i l pathfinding. Therefore, the results obtained with laminin-nidogen blocking reagents are specific to that site.  Discussion Laminin is expressed uniformly in the basal lamina of the developing limb bud during the time that several neuronal projections are established, including the T i l pioneer neuron pathway. As potential mediators of laminin induced neuronal outgrowth or guidance, (31 containing integrins were found on T i l growth cones, axons and cell bodies. Disruption of a conserved nidogen binding site on the y chain of laminin during development of the T i l pathway disrupts pioneer neuron pathfinding. Surprisingly, the T i l growth cones cease proximal extension, at a precise region within the limb, where they typically make a ventral turn. Extending the length of the culture period does not overcome the effect. Localization of stalled growth cones indicates that they stall within proximity of guidance cues found in the trochanter limb segment, and in the case of secreted semaphorins, these cues are properly localized. In addition, the basal lamina is not disrupted in these embryos.  Potential roles of nidogen When the nidogen recognition sequence on laminin is blocked, T i l axons stall at the location of the ventral turn in the trochanter limb segment, which is essential to their continued migration in to the CNS. Since laminin is evenly expressed in this region, it is  112  not likely that laminin is a chemoattractant that directs axons ventrally, nor is it likely to be a chemorepulsive agent that repels axons from the dorsal quadrant. Why does disrupting the nidogen binding site on laminin perturb axon pathfinding? The even laminin distribution is consistent with a permissive role of laminin. However, in these experiments, antibodies and peptides that were designed to disrupt laminin-nidogen interactions were used. Therefore the defects observed could be due to disruption of nidogen function rather than laminin function. Unfortunately, the expression pattern of nidogen in the developing embryo is unknown and the possibility that nidogen is discretely expressed in the developing limb bud cannot be discounted. Discrete localization of nidogen could guide Ti 1 neurons directly. Alternatively, nidogen could act in concert with another guidance cue to facilitate the steering event. K i m and Wadsworth (2000) have demonstrated that nidogen is essential for pathfinding of neurons in C. elegans, and may modulate the effect of other guidance cues such as netrins. They suggest that the effect of nidogen mutations is directly due to the loss of nidogen as opposed to secondary effects on basal lamina stability since the basal lamina of nidogen mutant animals is intact. Thus nidogen has important guidance activity and could be the sole mediator of the stalled Ti 1 axon effect. Since the antibodies and peptides used in this study were designed to disrupt basal lamina interactions, and were found to disrupt the integrity of the basal lamina in vitro (Kadoya et al., 1997) it was essential to determine the structural integrity of the basal lamina in laminin-nidogen blocked embryos. Several lines of evidence suggest that the basal lamina integrity is unaffected in laminin-nidogen blocked embryos. Enzymatic digestion of the basal lamina in grasshopper embryos (which completely removes the  113  basal lamina) results in severe morphological changes in the limb bud as well as retraction of T i l axons due to loss of adhesion to the basal lamina (Condic and Bentley 1989 a,c). This was not observed in laminin-nidogen blocked embryos. Additionally, we demonstrated that laminin and Semaphorin 2a distribution (at the light microscopic level) was unaffected in laminin-nidogen blocked embryos. Thus from these observations we conclude that blocking laminin-nidogen using antibodies and peptides causes a discrete and specific molecular lesion that does not compromise the integrity of the basal lamina or the localization of other secreted molecules. Furthermore, blocking a cell adhesion site on the beta chain of laminin that is involved in modulating netrin activity has no effect on T i l pathfinding (Hopker et al., 1999) Therefore, the defects observed with laminin-nidogen blocking are a direct result of blocking this site and not a general effect of blocking laminin or a secondary effect of disrupting basal lamina interactions.  The role of basal lamina in neuronal pathfinding We have shown a surprising role of laminin in neuronal pathfinding. Previous work by Condic and Bentley (1989c) had suggested that an adhesive role for the basal lamina, when they found that enzymatic removal of the basal lamina resulted in decreased neuronal adhesion to the substratum and axon retraction to the cell bodies (Condic and Bentley, 1989a). However, after retraction, T i l cell bodies could extend and pathfind in the absence of a basal lamina (Condic and Bentley, 1989a). In the present study, a very specific molecular interaction within the basal lamina was disrupted, whereas Condic and Bentley (1989a) removed the entire basal lamina using various enzymes such as elastase. Immunofluorescence with laminin / antisera of peptide blocked embryos indicates that blocking laminin-nidogen interactions does not disrupt  114  the basal lamina. Enzymatic removal of the basal lamina may result in a compensatory mechanism within T i l growth cones, such that they no longer depend on the basal lamina for adhesive contacts (Condic and Bentley, 1989c). T i l axons were found to adhere to the epithelium in the absence of the basal lamina, where before they were dependent on the basal lamina (Condic and Bentley, 1989c). This indicates that the neurons are capable of compensating for the lack of basal lamina interactions perhaps by down regulating basal lamina receptors like integrins and up regulating cell adhesion molecules, such as C A M ' s . In this study, disrupting the laminin-nidogen interactions could perturb expression of growth cone receptors, such as integrins, without affecting expression of other molecules, such as C A M s . Therefore, i f a compensatory mechanism within T i l growth cones accounts for proper T i l pathfinding in the absence of basal lamina, as in the Condic and Bentley studies, this mechanism may not have been activated in the lamininnidogen block.  Growth cone priming is essential for pathfinding In this study, we have demonstrated a requirement of laminin-nidogen in neuronal pathfinding. When laminin-nidogen is blocked with antibodies or peptides, T i l axons cease growth in the trochanter limb segment, at the site of a ventral turn. Prolonged culturing of these embryos does not rescue the stalled axons, indicating that the velocity of neuronal growth was not a factor. Analysis of laminin and Semaphorin 2a expression indicated that the distribution of these two molecules was unaffected by the blocking procedure, ruling out the possibility that the basal lamina suffered gross structural defects. Although the expression of nidogen was not determined in this study, laminin is found evenly distributed in the basal lamina, suggesting a permissive role for this basal  115  lamina molecule. How does blocking a nidogen recognition sequence on laminin effect T i l pathfinding? The pathfinding of neuronal growth cones relies on several factors, which can be classified as instructive or permissive. The growth cone is a dynamic motor apparatus, extending and retracting, responding to the environment integrating not only instructive cues, but also permissive cues. This assures a balance of adhesion to the substratum and motility to guarantee growth cone responsiveness. If this balance is disrupted, the growth cone would not be able to respond to instructive cues, resulting in pathfinding errors. Hopker et al. (1999) have demonstrated that the laminin can alter the guidance activity of the laminin-related netrin family of molecules, suggesting that the simultaneous presentation of instructive and permissive cues is pertinent to the effect of guidance molecules. This may come about by direct binding of netrins and laminin which may ensure a certain conformation that would preferentially activate one netrin receptor over another. In the developing limb bud, laminin has the potential to modulate the activity of several guidance molecules, due to its ubiquitous and even distribution in the basal lamina of the developing limb bud during axon outgrowth. Thus in this capacity, laminin could be an important co-factor to a guidance molecule such as the inhibitory protein Semaphorin 2a, which is required for the ventral turn in the trochanter limb segment (Isbister et al, 1999). However, the abnormalities observed with laminin perturbation were distinct from those resulting from Semaphorin 2a blocking. For example, T i l axons did not explore areas that highly express Semaphorin 2a, which they normally avoid. Therefore, another mechanism could account for the observed defects in axon pathfinding when laminin is blocked.  116  Since the growth cone is in constant contact with the environment, sampling and responding to many molecular cues, disruption of laminin-nidogen interactions in the immediate environment of the Ti 1 growth cones could have severe effects on the molecular constituents found in the growth cone. Thus while blocking laminin-nidogen interactions has little effect on axon outgrowth, (Til axons extend proximally to the CNS), it has severe effects on pathfinding since T i l growth cones are incapable of making a steering decision that involves multiple cues (Kolodkin et al., 1992; Isbister et al., 1999; Wong et al., 1999; Bonner and O'Connor, 2000). We propose that rendering laminin-nidogen inaccessible to the T i l growth cones affects laminin receptors such as integrins. It has recently been demonstrated that integrin levels in neuronal growth cones can be rapidly modulated based upon availability of both laminin and aggrecan in vitro (Condic and Letourneau, 1997; Condic et al., 1999) implicating integrins as mediators of the balance between adhesion to the substratum and growth cone motility. Condic and Letourneau (1997) have established that when neurons are plated on low laminin concentrations, integrin receptors increase on the growth cones, resulting in increased adhesion to various substrates, suggesting that neurons can rapidly and dynamically respond to their environment and maintain a balance between adhesion and motility. When applied to our system, blocking laminin in the developing embryo could influence integrin receptors on the growth cone, thereby affecting adhesion to the substratum. This could effect a steering decision in the trochanter, however does not appear to affect motility before the trochanter.  117  Why is motility inhibited at the ventral turn and not before? The proposed effect on integrins or another receptor on the growth cones may not be evident before the trochanter since the axons do not turn until they reach the trochanter. Growth conesubstrate adhesive contacts are sufficient to generate the force required to dictate direction of neuronal growth in vitro (reviewed by Suter and Forscher, 2000). While an instructive guidance cue may be signaling the growth cone with respect to the directionality of a turn, the permissive machinery (through laminin and integrins, for example) may be required to generate the asymmetric cytoskeletal rearrangements regardless of the nature of the instructive cue. Therefore, instructive guidance cues are present, but in the absence of permissive cues, such as laminin, these cues cannot result in growth cone turning (figure 23). As they migrate, growth cones encounter different cues that are spatially and temporally regulated. The exact sequence of encountered cues may be critical for the growth cone to appropriately respond to upcoming guidance information suggesting that the growth cone retains information, perhaps in the form of modulated receptor subtypes (Dodd et al., 1988; Shirasaki et al., 1998; Brose and Tessier-Lavigne, 2000; Diefenbach et al., 2000). Since the ventral turn of the T i l neurons involves multiple cues, all of which are not detected in laminin-nidogen blocked embryos, we speculate that permissive cues are responsible for priming the growth cone with adhesive receptors such as integrins that facilitate axon pathfinding.  118  Figure 23: Proposed mechanism of laminin in axon guidance. (A) Schematic representation of the T i l pathway in unperturbed grasshopper embryos. Laminin is expressed evenly in the basal lamina (green) by hemocytes (green cells). (B) In lamininnidogen blocked embryos, T i l axons stall at the site of a steering decision. (C) Diagram of a migrating neuron with growth cone responding to attractive (+) cues. Microtubules are found in the axon shaft and actin filaments are found in the growth cone. The inability of T i l growth cones to make a steering decision could result from decreased adhesion in conjunction with steering decisions. (D) Guidance molecules bind to guidance receptors which could be directly or indirectly linked to laminin receptors, such as integrins. In the presence of laminin, the combination of guidance receptor activation and laminin binding to laminin receptors mediates growth cone turning. (E) When laminin is blocked, the loss of laminin receptors or the inability of receptors to bind to laminin could result in insufficient adhesion, and a decreased coupling to the actin cytoarcheticture which normally enables the turning response. Ti, tibial limb segment, Fe, femur limb segment, Tr, trochanter limb segment, Cx, coxal limb segment.  119  \20  CHAPTER 4: SUMMARY AND CONCLUSION The mechanisms underlying axonal extension and guidance have been described using both cell biological and genetic approaches. These analyses have provided the field with a variety of guidance molecules and receptors and their potential linkages to the highly dynamic actin cytoskeleton within the growth cone. Guidance molecules and receptors provide the migrating neurons with directional cues so that migration to the target can be achieved. These molecules can act as attractive or repulsive cues and in some cases can be bifunctional depending on a number of conditions. In this study, the role of laminin, a basal lamina molecule constitutively expressed during pioneer axon outgrowth was assessed. Unlike guidance molecules that have a restricted distribution and cause a stereotyped turning response either away from or toward the source of the cue, laminin represents a permissive cue that is evenly distributed and that T i l axons neither migrate towards nor avoid. In spite of this, laminin has proved to be essential for axon guidance. Not affecting axon outgrowth, laminin has a profound effect on a steering decision that involves multiple guidance cues, including members of the semaphorin family (Bonner and O'Connor, 1999). In addition, this study has highlighted a novel mode of basal lamina deposition and underscores the importance of basal lamina interacting molecules and transmembrane receptors for basal lamina assembly.  121  In the initial characterization of laminins in grasshopper, the (3 and / chains of laminin were cloned. The deduced amino acid sequence of (3 and / chains of grasshopper laminin contained conserved functional motifs that are involved in netrin signaling and cell adhesion (YAGSR) and nidogen binding (DPNAV) (Graf et al., 1987; Poschl et a l , 1996; Hopker et al., 1999). Subsequence analysis of these clones demonstrated that grasshopper laminins are likely to have the same tertiary structure as other laminins. Therefore, grasshopper laminins are probably assembled into a trimer, secreted, and polymerized into a matrix within the basal lamina. A temporal study of laminin expression revealed that it's expression is coincident with the outgrowth of T i l pioneer neurons in the periphery and M P pioneer central neurons. Laminin is expressed by hemocytes of mesodermal origin throughout the embryo. As the basal lamina lays directly beneath the epithelium, it was surprising to find that laminin was not found in the epithelium throughout the embryo. The random distribution of laminin expressing hemocytes as well as their filopodia and lamellipodia suggested that these cells were migratory. This was confirmed with time-lapse imaging, and it was found that hemocytes migrate in random directions throughout the limb bud. The filopodia and lamellipodia extend in the direction of the migration, and can extend from any part of the cell. Thus, the trailing edge of the hemocyte can become the leading edge, as cells were commonly found to reverse direction. Like the directionality of migration, the velocity was also variable. Immunofluorescence of laminin revealed an even distribution of laminin in the basal lamina at all time points examined the earliest coinciding with T i l axon outgrowth. The even distribution of laminin does not reflect the structure of the basal lamina as a  122  whole, as electron microscopic analysis demonstrated a basal lamina that changes in thickness temporally and spatially (Anderson and Tucker, 1989). The even distribution of laminin in the basal lamina is achieved by migrating hemocytes. In addition, another component of the basal lamina, the antigen recognized by the 3H12 antibody, is also expressed by the same migrating hemocytes. Once laminin is secreted from the hemocytes, other tissues that are stationary, for example muscle pioneers or the epithelium probably mediates the polymerization of laminin into an orthagonal array. We have demonstrated that (31 integrin is expressed on the epithelium of the developing limb bud and suggest that p i integrins are, in part, responsible for the correct localization and polymerization of laminin once it is secreted. Furthermore, hemocytes probably do not play a role in laminin polymerization or basal lamina assembly, but act solely to secret components of the basal lamina. The even expression of laminin in the basal lamina also has implications for axon guidance. During the time that the T i l axons are extending into the CNS, laminin is evenly expressed in the basal lamina. If we define instructive cues as discretely localized molecules that effect the directionality of axon growth, then laminin cannot be classified as an instructive cue. Instead, laminin is evenly expressed, and does not appear to affect the directionality of pioneer neuron growth. Therefore, it is classified as a permissive cue. As they are migrating, the T i l growth cones encounter laminin along the way. Laminin amounts remain the same during the entire T i l pathway, although availability of other cues, for example semaphorins, changes as the growth cone continues toward the CNS (Bonner and O'Connor, 1999). Laminin could merely be providing a substrate for  123  migration, a signal that communicates to the neuron to continue migrating. If this were so, then neurons would not be capable of migration when laminin function is blocked. This study examined the function of two distinct sites on the laminin molecule. Antibodies and peptides that acted as competitive inhibitors disrupted these sites. The first site, the conserved cell adhesion site (YAGSR) that is involved in netrin signaling, had no effect on the pathfinding of the T i l pioneer neurons. Neither antibodies nor peptides could alter the course of these neurons, even at high concentrations. In contrast, antibodies and peptides that are specific to the nidogen recognition motif (DPNAV) on the / chain of laminin had profound effects on the course of the T i l neurons. In the presence of either antibodies or peptides that were designed to perturb the binding of nidogen to laminin, T i l axons ceased growth. Prolonged culturing of blocked embryos did not overcome the effect of stalled axons and the stalled growth cones were localized to the trochanter limb segment, the site where the axons normally turn ventrally. T i l axons rarely stalled before the trochanter, and were never found to have initiated the ventral turn. Even in embryos that were slightly older at the onset of the experiment (and therefore had axons that had extended into the proximal tibia or distal femur) T i l growth cones still ceased growth at the trochanter, suggesting that the effect of the blocking reagents occurred swiftly after application. Therefore, we conclude that the reason that T i l axons stall in the trochanter is not due to an inhibition of growth, because the T i l axons grow along the tibial and femur basal lamina in which laminin has been blocked without any abnormalities. In addition, the integrity of the basal lamina was tested with immunofluorescence and found to be intact in laminin-nidogen blocked embryos.  124  If blocking the putative nidogen recognition sequence results in nidogen release from the heterotrimer, then nidogen may be degraded by proteases (Mayer et al., 1993b). Localization of nidogen before and after laminin-nidogen has been blocked would address this question (see below). Laminin signaling is mediated through the integrins, and (31 integrin was found to be expressed along T i l cell bodies, axons and growth cones. Although (31 integrins can bind to several sites on the laminin heterotrimer, thus far there is no (31 integrin binding site in the vicinity of the nidogen binding site on the y chain. Therefore there is no evidence that p i integrins could be mediating this effect, although this is something that can be tested in the future (see below). We propose that laminin-nidogen, acting through a receptor on the growth cone, whether integrin or not, is equipping the growth cone to respond to upcoming cues. By altering laminin-nidogen in the environment, the growth cone is no longer able to respond to other environmental cues such as semaphorins. It is possible that by disrupting laminin-nidogen we could also be disrupting the localization of semaphorins, although semaphorin 2a expression appeared to be unaltered in laminin-nidogen blocked embryos. Therefore, blocking laminin-nidogen could be uncoupling instructive and permissive cues.  Future  studies Blocking laminin-nidogen results in the inability of T i l axons to negotiate a  steering decision. The results of this study lead to intriguing questions. For example, what is the role of nidogen? Furthermore what receptors on the growth cone and what  125  intracellular signaling events may be mediating laminin-nidogen signaling? The following proposed experiments would address these questions.  Nidogen localization The striking result of laminin-nidogen perturbation, stalled axons, could result from the disruption of laminin function, or from the disruption of nidogen. When nidogen is released from laminin, it is more sensitive to cleavage by proteases (Mayer et al., 1993b). Therefore, by disrupting the binding of nidogen to laminin, nidogen could be degraded. The localization of nidogen could be determined using immunofluorescence with nidogen specific antibodies. In another approach, the peptides used to disrupt laminin nidogen complexes, which bear the sequence D P N A V , could be labeled with a fluorophore and applied to intact embryos (Molecular Probes). If specific, the peptide should only bind areas of the limb basal lamina that contain nidogen. In addition, the GST-fusion protein that contains the peptide sequence D P N A V can also be applied to the embryo in the same manner, and then reacted with anti-GST antibodies. In addition to localizing nidogen in intact embryos, the same reagents could be used in laminin-nidogen blocked embryos to determine i f nidogen is degraded when the interaction with laminin is blocked. These approaches should provide useful information with regard to nidogen localization during T i l axon outgrowth.  Integrins If blocking laminin-nidogen results in alterations of integrins, perhaps by down regulation, then immunofluorescence of integrin receptors on the growth cone in a laminin-nidogen blocked embryo may be informative. Integrin immunofluorescence on  126  limb fillets of grasshopper embryos has been successful, and using these antibodies to label integrins after culturing is plausible. In addition, to determine if integrins are mediating laminin responses in T i l growth cones, embryos could be cultured in the presence of an a6 integrin blocking antibody (Pharmagin) and the (31 integrin antibody that was used in this study.  LAR The L A R family of receptor tyrosine phosphatases was shown to bind to lamininnidogen complexes (O'Grady et al., 1998). Perturbation of L A R phosphatase activity in Drosophila and leech results in pathfinding defects, including stalled or shortened axonal processes similar to results obtained here with laminin nidogen complexes (Desai et al., 1997; Gershon et al., 1998; Baker and Macagno, 2000a; Baker and Macagno, 2000b). Thus there is a distinct possibility that laminin-nidogen signaling is mediated by L A R tyrosine phosphatases. To address the possibility that laminin-nidogen may be acting through receptor tyrosine phosphatases, the localization of L A R receptors on T i l growth cones and axons could be conducted with antibodies specific for the leech L A R receptor. To determine if phosphatase activity is required for laminin-nidogen signaling, phosphatase inhibitors could be applied to grasshopper embryos undergoing axon extension. Vanadate, a non-specific phosphatase inhibitor promotes neurite outgrowth from cultured PC-12 cells (Rogers et al., 1994), indicating that this inhibitor is effective on neuronal cell types.  127  Concluding remarks In conclusion, work in this thesis has resulted in some interesting findings with respect to basal lamina assembly and axon guidance. The literature has for some time indicated the importance of cell-surface laminin receptors in mediating laminin polymerization and basal lamina assembly (Colognato and Yurchenco, 2000). This study has indicated that the deposition of laminin and possibly others into the basal lamina can be accomplished by cells that have no fixed location, but migrate randomly, indicating other tissues in the organization of the basal lamina. Although spatially distinct and separated by the basal lamina, distinct tissue types may act in concert to ensure the proper assembly of the basal lamina. 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