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Mechanisms underlying neuronal growth cone pathfinding Isbister, Carolyn Marie 1999

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MECHANISMS U N D E R L Y I N G N E U R O N A L GROWTH CONE PATHFINDING by C A R O L Y N MARIE ISBISTER B . S c , Simon Fraser University, 1993 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Program in Neuroscience We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A July, 1999 © Carolyn Marie Isbister, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT From the initial stages of axon outgrowth to the formation of a functioning synapse, growth cones of developing neurons integrate and respond to multiple environmental guidance cues. To explore growth cone guidance mechanisms in vivo, we have taken advantage of the ease of access and manipulation of the well-characterized model system, the developing grasshopper embryo. To elucidate the role of substrate adhesivity in growth cone pathfinding, we developed an in vivo assay for measuring filopodial-substrate adhesivity using the T i l pioneer neuron pathway of the embryonic grasshopper limb. Using time lapse imaging and a combination of rhodamine-phalloidin injections and D i l labeling, we demonstrate that filopodial retraction rate following treatment with cytochalasin D or elastase reflects the degree of filopodia-substrate adhesivity. There is no difference between retraction rates of filopodia extending towards the correct target and filopodia extending away from the correct target. The homogeneity in filopodial retraction rates, even among turning growth cones where differential adhesivity might be expected to be greatest, strongly indicates that differential adhesion does not govern T i l pioneer neuron pathfinding. In addition to contact-mediated mechanisms, secreted molecules, such as semaphorins, can also steer growth cones. We demonstrate that a novel secreted semaphorin in grasshopper, gSema 2a, functions as a chemorepulsive guidance molecule critical for T i l pioneer axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the T i l axon projection. Simultaneous perturbation experiments indicated that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo. Furthermore, Sema 2a is expressed in two overlapping perpendicular gradients: a steep distal-proximal and a shallow dorsal-ventral gradient. Although the T i l growth cone will sample both the steep and shallow gradients, the majority of pathfinding errors occur within the shallow dorsal-ventral gradient. Although the gradient steepness differs, the absolute levels of Sema 2a at the stereotyped growth cone decision points are comparable. Ourtresults indicate that it is the steepness, or shape, of the gradient that encodes the degree of chemorepulsion. Finally, using these gradient data we propose theoretical mechanisms of growth cone gradient detection in vivo. i i i T a b l e o f C o n t e n t s A B S T R A C T i i T a b l e o f C o n t e n t s i v L i s t o f F i g u r e s v A c k n o w l e d g e m e n t s v i i I. I N T R O D U C T I O N 1 I I . F I L O P O D I A L A D H E S I O N D O E S N O T P R E D I C T G R O W T H C O N E S T E E R I N G E V E N T S IN VIVO. 19 Introduction 20 Materials and Methods 22 Results 30 Discussion 53 I I I . D I S C R E T E R O L E S F O R S E C R E T E D A N D T R A N S M E M B R A N E S E M A P H O R I N S I N N E U R O N A L G R O W T H C O N E G U I D A N C E IN VIVO 59 Introduction 60 Materials and Methods 63 Results 69 Discussion 96 I V . G R A D I E N T S H A P E E N C O D E S G R O W T H C O N E G U I D A N C E I N F O R M A T I O N IN VIVO 102 Introduction 103 Materials and Methods 105 Results 109 Discussion 139 V . G E N E R A L D I S C U S S I O N 143 The role of adhesion in growth cone guidance. 143 The role of Semaphorins in Til pioneer axon guidance. 145 The balance of multiple signals guides neuronal pathfinding. 148 Axon guidance by gradients of chemorepulsion. 150 Growth cone gradient-reading mechanisms. 152 How are chemotactic gradients translated into directional growth cone migration? 154 Summary of possible mechanisms guiding the Til pioneer projection. 155 Conclusion 156 V I . R E F E R E N C E S 157 iv List of Figures Figure 1. Schematic of Til pioneer neuron pathway 25 Figure 2. Categorization of growth cone filopodia as on-axis or off-axis 27 Figure 3. Cytochalasin D disrupts internal cytoarchitecture of Til filopodia. 31 Figure 4. The majority of filopodia eventually retract after cytochalasin D treatment. 34 Figure 5. Filopodial retraction rate following cytochalasin D or elastase application accurately reflects in vivo substrate adhesion 36 Figure 6. Pathfinding behaviour of non-turning Til growth cones before and after treatment with cytochalasin D 39 Figure 7. Pathfinding behaviour of a Til growth cone turning toward the Cxi cells before and after treatment with cytochalasin D 44 Figure 8. In vivo Til growth cone pathfinding behaviours and filopodial extension rate are not governed by differential filopodial substrate adhesion 47 Figure 9. Elastase experiments confirm that in vivo filopodial extension rate and pathfinding behaviours of Til growth cones are not governed by differential substrate adhesion. 49 Figure 10. Amino acid comparison of gSema 2a and dSema 2a 70 Figure 11. Recombinant peptides generated for antibody production, Western and Northern blot analysis 72 Figure 12. Sema 2a is expressed in the developing limb bud during Til pioneer axon outgrowth 75 Figure 13. Sema 2a and Sema la expression during Til pioneer axon outgrowth 77 Figure 14. Antibodies directed against the semaphorin domain of Sema 2a disrupt Til pioneer pathfinding. 81 v Figure 15. Summary of Sema 2a antibody perturbation experiments 84 Figure 16. Sema 2a and Sema la act in combination to guide Til axon pathfinding. 88 Figure 17. Summary of combined Sema 2 a and Sema la antibody perturbation experiments.. 91 Figure 18. Relationship of the Til projeciton in control and semaphorin antibody blocking experiments 94 Figure 19. Sema2a protein expression during the Til pioneer projection into the CNS. 110 Figure 20. Sema 2a gradient analysis 112 Figure 21. Sema 2a is expressed in exponential distal-proximal and dorsal-ventral gradients. 115 Figure 22. Laminin protein expression within the grasshopper limb bud at 31% embryonic development. 119 Figure 23. Til pioneer growth cones occasionally misproject up the chemorepuslive gradients of Sema 2a .*. 122 Figure 24. Steep distal-proximal gradient of Sema 2a ensures Til pioneer axonogenesis and proximal outgrowth 124 Figure 25. Growth cones emerging from the distal pole of the distal Til cell body reorient immediately to migrate down the Sema 2a gradients 129 Figure 26. Til pioneer growth cones use the fractional change mechanism of gradient-reading to detect the steepness of the exponential gradients of Sema 2a 134 vi Acknowledgments I thank my supervisor, Dr. Tim O'Connor, for his excellent support and for allowing me the freedom to pursue my own research interests under his guidance. Tim has been a great sport, he has graciously offered himself to the lab as a target for our numerous good-natured barbs and he invariably picked up the tab for beer and pool at the pub. I thank Jennifer Bonner, whom I have had the pleasure to work with over the last three years. Her clarity of thought and scientific insight has proved crucial for many of the experiments described in my thesis. In addition to our many stimulating discussions on science and beyond, Jen has been a great friend and colleague. Thanks to Oliver Prange, who has an uncanny ability to spark my argumentative nature, yet he always leaves me laughing and with a lightened heart. I must also express my gratitude to Dr. Alaa El-Husseini who, during the first two years of my PhD, held my hand and answered my endless questions. I acknowledge Dr. Tim Murphy for the use of his confocal microscope and computers. Several colleagues made significant contributions to my thesis and I would to thank them: Dr. Paul Mackenzie for advice on the statistical analysis in Chapters 2 and 4, and for the confocal microscopy and curve fit analysis described in Chapter 4. Mr. Arthur Tsai and Dr. Alex Kolodkin for the cloning and sequencing of gSema 2a described in Chapter 3. Lastly, I thank my family for their unquestioning understanding and continued support over the years. And most importantly, I thank Paul. His passion and intensity push me to strive, challenge my thoughts, fuel my dreams and make my life rich and sweet. I. INTRODUCTION A central issue in neuroscience is how developing neurons establish correct connections with their targets. The formation of precise neuronal connectivity can be thought of as a series of events, beginning with axon initiation and directed neurite outgrowth. Target recognition and, finally, refinement of the contact in synapse formation follow this initial period of neuronal pathfinding. The processes of neuronal development do not stop at birth, but continue throughout the lifetime of the organism as the underlying mechanisms of learning, memory and neuronal regeneration. Typically, neuronal pathfinding and target recognition rely on molecules in the environment for guidance and occur before neurons are functionally active, thus these mechanisms are considered activity-independent. In contrast, synapse specificity and refinement are classically considered to be dependent on neuronal activity. Recently, however, activity dependent and independent mechanisms of neuronal circuitry formation have become less distinct. Molecules typically considered to function in pathfinding have been shown to regulate synaptic plasticity (Martin and Kandel, 1996; Schuster et al., 1996a; Schuster et al., 1996b), and neural activity has been shown to be necessary for the initial targeting decisions made by pathfinding axons (Catalano and Shatz, 1998; Crair, 1999). Each stage of neuronal connectivity is, in part, dependent upon the coordinated expression of many cues in the environment, and the detection and integration of these signals by the expanded motile tip of the developing axon, the growth cone (Kater and Rehder, 1995; Goodman, 1996; Tessier-Lavigne and Goodman, 1996). The neuronal growth cone is a highly dynamic sensory-motile device, continuously reading the molecular terrain and transducing the information into directed motility. Although more than a century has passed since Ramon Y Cajal first implicated growth cones in neuronal pathfinding, the mechanisms governing this 1 fundamental developmental process remain unclear. Thus, it is the aim of this thesis to investigate some of the mechanisms that underlie neuronal growth cone pathfinding in vivo. Embryonic grasshopper as a model system Investigation into how environmental guidance molecules interact with growth cones to direct pathfinding within the developing organism is facilitated by an experimental system that allows the observation and analysis of neuronal growth cones as they pathfind in vivo. The development of the embryonic grasshopper limb bud suits these requirements. The first peripheral projection to the CNS within the grasshopper embryo is established by a pair of sibling pioneer neurons, which emerge from the underlying epithelium at the tip of the developing limb bud. These neurons, termed the tibial (Til) pioneers, arise from a single mother cell at about 30% of embryogenesis and complete their projection into the CNS by 35%, typically over a period of 24 hours. The development of the T i l pioneer projection in the embryonic grasshopper offers several experimental advantages for investigating the molecular and cellular events of neuronal growth cone guidance in general. First, of particular experimental advantage is the ability of embryonic grasshoppers, once removed from their egg and amnion, to develop normally in culture media for several days. This provides an experimental system that allows for the observation, manipulation and analysis of growth cones as they pathfind in vivo. Secondly, the development of the T i l pioneer neurons is a simple and stereotyped system that has been studied extensively with many of the guidance molecules encountered by the T i l pioneer growth cones previously identified (Bentley and O'Connor, 1992; Sanchez et al., 1995). Consequently, this projection has proven to be a useful model for the analysis of several aspects of neuronal pathfinding in vivo, including the role of pioneer neurons, the nature of growth cone guidance information, and the mechanisms growth cones 2 employ to steer in response to such information. In addition, pioneer neurons are not specific to the embryonic grasshopper, these specialized neurons have been found in a broad variety of organisms including fruit fly, fish, and mammals (Jacobs and Goodman, 1989; McConnel et al., 1989; Pike et al., 1992). Thirdly, sequence analysis has demonstrated extensive homology between many of the vertebrate and insect neuronal growth cone guidance and adhesion molecules. For some of these molecules, the remarkable sequence similarity also extends to the spatial and temporal pattern of expression (see reviews by Mark et al., 1997; Chien, 1998). Finally, it is becoming apparent that the extensive homology between invertebrate and vertebrate neuronal guidance molecules is reflected functionally (Goodman, 1994; Luo et al., 1997; Mark et al., 1997; Chien, 1998); therefore, the fundamental mechanisms of axon guidance and extension are shared between invertebrates and vertebrates. Following the completion of the T i l pioneer projection into the CNS, additional peripheral pioneer neurons arise throughout the elongating grasshopper limb bud and also extend axons along specific routes. These pioneer axons fasciculate into bundles and establish the pattern of afferent nerve trunks along which later projecting sensory growth cones migrate. Shortly after establishing the connection with the CNS, the T i l pioneer axons lose their attachments to the epithelium and move through the basal lamina, becoming displaced into the interior of the limb where they subsequently die (Kutsch and Bentley, 1987). The requirement of the transient T i l pioneer projection for the accurate pathfinding of follower neurons is variable. Subsets of neurons display the ability to pathfind successfully into the CNS in the absence of the pioneer pathway, demonstrating that these follower neurons are also endowed with the appropriate machinery to detect and integrate the same guidance cues the pioneer growth cones encounter (Keshishian and Bentley, 1983). In contrast, populations of neurons that arise distal to the T i l pioneer cell bodies cannot pathfind into the CNS without the presence of the pioneer scaffold (Klose and Bentley, 1989). Pioneer neurons, therefore, establish early pathways when 3 embryonic distances are short and the environment is relatively simple. When the embryonic environment becomes more complex, these pioneer axons provide distinct pathways for later developing neurons to follow. The experiments described in this thesis employ the T i l pioneer projection of the developing grasshopper limb bud as a model system for the study of neuronal growth cone pathfinding mechanisms in vivo. Cytoskeletal dynamics underlying growth cone motility and steering Filopodia and lamellipodia are the structures that extend and retract from the growth cone body to explore the local environment and direct steering. Growth cone steering can be broken down into a series of sequential events, beginning with growth cone exploration and identification of target-directed filopodia (termed site selection), followed by stabilization of target-directed filopodia and, finally, axon formation (Tanaka and Sabry, 1995). Growth cone shape changes during pathfinding are preceded by reorganization of the cytoskeleton; thus, cytoskeletal dynamics are believed to predict subsequent growth cone steering events. Therefore, although it is uncertain how growth cones mediate steering events during pathfinding, it is accepted that the cytoskeleton plays a central role. The cytoskeleton, composed of actin microfilaments, microtubules and intermediate filaments, is a major constituent of neurites. Each type of filament has a characteristic spatial distribution within neurons: parallel arrays of intermediate filaments are within neurites but not in growth cones; microtubules appear bundled together in the axon shaft and extend into the growth cone body, they may also extend into the growth cone periphery; and actin is found at the leading edge of the growth cone and exclusively comprises the cytoskeletal network of filopodia (Suter and Forscher, 1998). The majority of research has focussed on the roles for 4 actin and tubulin, as these cytoskeletal components have been implicated in many critical axonal guidance events. Intermediate filaments, however, are not considered to play a major role in the initial elaboration and stabilization of growing axons (Phillips et al., 1983; Shaw et al., 1985). Actin dynamics in growth cone motility and steering. The localization of actin primarily within the highly motile lamellipodia and filopodia has focused attention on the role of actin in growth cone steering events (Tanaka and Sabry, 1995; Mitchison and Cramer, 1996). At the core of each filopodium is a bundle of actin filaments that extend into the lamella. In lamella long actin filaments criss-cross forming a meshwork beneath the growth cone periphery. The actin filaments are polarized in both filopodia and lamella, with their plus ends oriented toward the leading edge and their minus ends toward the centre of the growth cone. Actin monomers are assembled at the plus end of filamentous actin (F-actin), and disassembled at the minus end. Leading edge advance of most motile cells is characterized by three kinetic processes involving actin: first, assembly of F-actin at the leading edge; second retrograde flow of F-actin networks, likely powered by myosin-based motors; and third, proximal recycling of F-actin in the region of the growth cone body in which microtubules and F-actin overlap. Given that increased monomer addition at the leading edge could advance the growth cone, the regulation of any or all of these processes would affect the rate of forward movement. For example, leading edge advance could be accelerated by increased assembly of F-actin at the leading edge, or slowing retrograde flow of F-actin, which would in turn enable increased monomer addition at the leading edge, or decreased proximal recycling of F-actin, which could also slow the rate retrograde flow and lead to increased monomer addition. Direct implication of actin dynamics in growth cone pathfinding has been demonstrated. Drugs that disrupt filamentous actin (F-actin) cause lamellipodial and filopodial collapse, and block the ability of neurons to accurately pathfind (Bentley and Toroian-Raymond, 1986; Chien 5 et al., 1993). Similarly, exposure of chick dorsal root ganglion growth cones to the inhibitory guidance molecule Collapsin-1 (chick secreted semaphorin) results in a loss of actin at the leading edge and collapse of the growth cone (Fan et al., 1993). Further studies by Fan and Raper (1995) demonstrated that dorsal root ganglion growth cones steer away from Collapsin-1 coated beads by a process involving lamellar and filopodial inhibition/collapse in the vicinity of Collapsin-1. In addition, experiments using cultured Aplysia neurons (Lin and Forscher, 1993) and embryonic grasshopper T i l pioneer neurons (O'Connor and Bentley, 1993) have demonstrated that during site selection, actin accumulates at the point where the growth cone contacts an attractive guidance cue and is depleted in the zone adjacent to it. It appears, therefore, that accumulation of F-actin in filopodia and growth cone branches accompanies continued growth, while reduction of F-actin accompanies withdrawal. Taken together, these studies suggest environmental cues may steer neurites by regulating local actin dynamics within the growth cone. Microtubule invasion of the target site is an early steering event. Directed neurite outgrowth is also dependent upon the regulation of microtubule dynamics. Microtubules are tightly bundled in the axon shaft and upon entering the central domain of the growth cone they splay out and continuously extend into and retract from the actin-rich peripheral domain (Tanaka and Kirschner, 1991). In growth cones, the more dynamic positive ends of microtubules are directed toward the periphery (Heidemann et al., 1981). This polarization allows microtubules to explore the periphery of the growth cone by randomly cycling between polymerization and depolymerization, a property called dynamic instability. Pharmacological studies using drugs which inhibit dynamic instability, yet do not eliminate microtubules within the growth cone, reveal that under these conditions axons will still grow, but cannot pathfind accurately. These results indicate that growth cone steering depends on the 6 dynamic microtubule ends (Tanaka et al., 1995; Rochlin et al., 1996). Specific microtubule invasion into the site of future growth during the early stages of steering has been observed in several systems (Sabry et al., 1991; Lin and Forscher, 1993; Tanaka and Kirschner, 1995). Sabry et al. (1991) have shown that during a turning event towards a discrete positive cue, microtubules in grasshopper T i l pioneer growth cones invade only the branch that becomes the nascent axon. During Ti 1 growth cone interaction with a boundary of less preferable substrate, however, microtubules will enter several branches before being stabilized in one. At present, the mechanism of microtubule site selection and stabilization is unclear; nonetheless it is accepted that for accurate growth cone pathfinding to occur, leading edge actin dynamics must be coordinated with central domain microtubule dynamics (Tanaka and Sabry, 1995; Suter and Forscher, 1998). The cytoskeletal-substrate coupling model for directed growth cone motility Recently, Lin and Forscher (1995) have demonstrated that contact mediated turning of cultured Aplysia neurons coincides with the slowing down of the retrograde flow of actin along the target interaction axis. In addition, microtubules were observed to preferentially extend into the region where retrograde actin flow was attenuated. From these findings a model for directed growth cone motility has arisen which proposes that when cell surface receptors bind extracellular matrix ligands, a multiprotein complex is recruited that links the receptor to the actin meshwork. This cytoskeletal-substrate coupling retards the retrograde flow of actin relative to the substrate, and, consequently, the continued assembly of actin at the leading edge results in leading edge advance. It is further proposed that the stabilization of the F-actin along the target interaction axis generates tension between the peripheral actin domain and the central microtubule domain, thereby promoting microtubule extension and formation of the nascent 7 axon. Thus, growth cones may use cell adhesion molecules and associated cytoskeletal binding proteins to regulate their extension rate and steering. Interactions between guidance molecule receptors and the cytoskeleton For target-directed outgrowth to occur, interactions between molecules that constitute guidance information and receptors on the growth cone surface must transmit signals to the interior of the growth cone that locally affect the organization and stability of the cytoskeleton. Many intracellular signalling mechanisms have been implicated in growth cone guidance, including intracellular Ca + 2 , cyclic nucleotides, tyrosine kinases and phosphatases, and the Rho family GTPases (Rehder etal., 1996; Luo et al., 1997; Viollet and Doherty, 1997; Van Vactor, 1998; Goldberg and Grabham, 1999; Hu and Reichardt, 1999). While there is evidence for the involvement of most second messenger systems in growth cone or cell motility, we will address only a selection of those signalling mechanisms implicated in growth cone motility. In support of the cytoskeletal-substrate coupling model of growth cone motility, members of cell adhesion receptor classes, such as Ig superfamily C A M S , cadherins and integrins, are capable of supporting axonal elongation in neurons and associate with the actin cytoskeleton (Gumbiner, 1993). The best characterized cell surface receptors with respect to signalling and cytoskeletal linkage are the integrins. Typically, integrins are heterodimers that mediate adhesion to various extracellular matrix molecules during cell migrations (Hynes and Lander ,1992). The components of integrin-associated cytoskeletal complexes have been analyzed mostly in focal adhesions, which consist of aggregated integrin receptors that link extracellular matrix components to actin stress fibres. Focal adhesions have been traditionally studied in cultured non-neuronal cells, where these contacts have been shown to contain a large number of structural proteins, such as talin and vinculin, as well as signal transduction proteins, such as 8 focal adhesion kinase (FAK), RhoA and src (reviewed by Hynes, 1992; Craig and Johnson, 1996). Recently, similar integrin-containing adhesion sites have been observed in neuronal growth cones and, furthermore, these neuronal contacts also associate with many of the same structural and signalling proteins (Renaudin et al., 1999). Another interesting cell-adhesion protein signalling mechanism involves heterophilic interactions between C A M S and the FGF receptor, a receptor tyrosine kinase. In a series of elegant studies, N C A M , L I and N-cadherin signalling has been shown to activate a FGF receptor-PLCy cascade (reviewed by Viollet and Doherty, 1997). Activation of the PLCy cascade could lead to PIP2 hydrolysis, generating IP3 and D A G , which can ultimately lead to changes in C a + 2 concentration. Interestingly, PIP2 is a well established modulator of actin binding protein gelsolin and profilin, which are inactivated when in a PIP2-bound state; thus, PIP2 hydrolysis could promote the release of gelsolin and profilin. Gelsolin is a calcium-activated severing protein, whereas profilin promotes plus end actin assembly, therefore, corelease of gelsolin and profilin could promote actin filament turnover, assembly and remodelling. The importance of tyrosine phosphorylation in axon guidance is supported by a considerable amount of evidence (reviewed by Friedman and O'Leary, 1996; Desai et al., 1997; Viollet and Doherty, 1997). Tyrosine phosphorylation can occur through stimulation of receptors with intrinsic tyrosine kinase activity or by non-receptor tyrosine kinases. Genetic analyses of various Drosophila receptor protein tyrosine phosphatases (RPTPs) and a receptor tyrosine kinase (RTK) have indicated that tyrosine phosphorylation may affect guidance by regulating growth cone adhesiveness and alter fasciculation and defasciculation events (Desai et al., 1997). Furthermore, two recent studies have demonstrated that tyrosine kinases and phosphatases act together to control the phosphorylation state of Enabled, a profilin associating 9 protein (Wills et al., 1999a; Wills et al., 1999b). These findings indicate that tyrosine phosphorylation can transmit signals from the cell surface to the actin cytoskeleton. Members of the Rho family of small GTP-binding proteins are also prime candidates for signalling agents that link extracellular guidance cues to the regulation of the actin cytoskeleton. Three members of this family have been shown to elicit distinct changes in the actin cytoskeleton; Rho stimulates the formation of stress fibers and focal adhesions, Rac stimulates the formation of lamella, and Cdc42 stimulates filopodial formation. Although the effects of Rho GTPases on the organization of the actin cytoskeleton are perhaps best characterized in fibroblasts, there is now compelling evidence of a similar role for these proteins in various aspects of axonal and dendritic outgrowth (Hall, 1998). For example, studies in Drosophila with dominant-negative and constitutively active GTPase mutants revealed motor and sensory neuronal growth cone pathfinding disruptions (Luo et al., 1997). In addition, Purkinje cells of transgenic mice expressing constitutively active Racl exhibited a reduction of axon terminals (Luo et al., 1996), and Rho and Racl modulate growth cone motility in cultured dorsal root ganglion neurons (Jin and Strittmatter, 1997). Interestingly, Racl appears to mediate dorsal root ganglion growth cone collapse following treatment with the secreted semaphorin, Collapsin-1 (Jin and Strittmatter, 1997). More recently, several downstream effectors of the Rho family proteins, such as the Cdc42 binding protein NWASP, have also been identified in neurons and shown to modulate actin assembly in vitro (Miki et al., 1996; Luo et al., 1997; Mik i et al., 1998; Rohatgi etal., 1999). Finally, it should be noted that every aspect of growth cone behaviour is likely to involve Ca signalling to some degree. Recently, Gomez and Spitzer (1999) have demonstrated that neuronal growth cones generate C a + 2 transients during migration in vivo, and furthermore, the rate of axon extension is inversely proportional to the frequency of transients. Some of the mechanisms by which alterations in C a + 2 within the growth cone may be translated into motility 10 changes include proteins associated with the regulation of cytoskeletal components, actin/myosin force-generating systems, and polarized vesicle fusion to the plasma membrane (reviewed by Sobue, 1993; Rehder et al., 1996; Goldberg and Grabham, 1999). Molecular mechanisms of growth cone guidance Early theories proposed that axon outgrowth is nonselective and diffuse; however, these theories have been largely rejected as a result of Sperry's evidence for specific neuronal recognition (Sperry, 1963). Sperry's chemoaffinity hypothesis proposed that axon guidance and target recognition are achieved by the matching of highly specific chemical affinities between individual neurons and their targets. Although activity-dependent mechanisms are also critical, particularly for the latter stages of neuronal connectivity, it is clear that there is a high degree of precision in growth cone pathway and target selection. Thus, the establishment of appropriate neuronal connections is, in part, dependent upon the coordinated interactions between pathfinding growth cones and their external environment. Over the past 15 years, considerable progress has been made in identifying and characterizing the molecules in the environment that interact with and provide guidance information to pathfinding growth cones. Neuronal guidance molecules are typically considered as either secreted into the local environment or cell surface associated, and further categorized as either attractive/permissive or repulsive for growth cone pathfinding. Diffusible molecules such as netrins, neurotrophins and secreted semaphorins may provide long distance growth cone guidance; whereas membrane-associated molecules such as the classic cell adhesion molecules likely support local, contact-mediated instruction to the growth cone (reviewed by Nieto, 1996; Goodman, 1996; Varela-Echavarria and Guthrie, 1997). 11 Short range, contact-mediated mechanisms of guidance Cell surface and extracellular matrix molecules regulate cell and substrate adhesion and include the classic cell adhesion molecules (CAMs), as well as other transmembrane or GPI-linked molecules (reviewed by Chiba and Keshishian, 1996). In general, contact-mediated guidance molecules can provide attractive signals, or alternatively, the trajectory of axons can also be directed by contact-mediated inhibition of advancing growth cones. For example, early studies demonstrated that growth cones of central neurons collapse when they contact peripheral nerves, and mature oligodendrocytes express proteins that inhibit axon extension (reviewed by Dodd and Jessell, 1988). More recently, contact inhibition, mediated by the Eph receptors and ephrins, has been demonstrated to influence retinal ganglion cell topographic mapping within the tectum (reviewed by O'Leary and Wilkinson, 1999). Therefore, contact-mediated mechanisms of guidance can promote attractive or repulsive events and thus play a central role in many aspects of neuronal development including axon extension, fasciculation, steering, target recognition and synapse stabilization (reviewed by Chiba and Keshishian, 1996). Grasshopper T i l pioneer growth cone pathfinding has also been shown to be dependent upon a variety of membrane-associated guidance molecules (Bentley and Caudy, 1983; Caudy and Bentley, 1986a; Kolodkin et. al., 1992; Sanchez et. al., 1995; Wong et. al., 1997). However, how these substrate-bound adhesion molecules interact with filopodia to direct growth cone steering events in vivo is not well understood. Evidence from early in vitro experiments indicated that growth cone filopodia may select the most adhesive pathway available for migration, supporting a model for growth cone steering based on differential expression of adhesive molecules in the environment (Letourneau, 1975). A n increase in filopodial-substrate adhesion of target-directed filopodia could lead to increased coupling of adhesion molecule receptors to the cytoskeleton and a differential increase in leading edge advance, thus steering the growth cone. In addition, previous studies have suggested that pioneer neurons in the 12 developing grasshopper limb are oriented by substrate-bound adhesion molecules organized in a distal-proximal gradient of adhesiveness (Caudy and Bentley, 1986b; Condic and Bentley, 1989; reviewed by Bentley and O'Connor, 1992). Thus, one of the goals of this thesis is to test whether growth cones are guided by differential adhesivity in vivo. In Chapter 2 we describe the development of an in vivo assay for measuring filopodial-substrate adhesivity using the well characterized T i l pioneer pathway of the embryonic grasshopper limb. Using this assay we examine whether filopodial-substrate adhesivity predicts growth cone steering events in vivo (Isbister and O'Connor, 1999). Long range, diffusible mechanisms of guidance * Additional guidance mechanisms distinct from adhesion are most clearly demonstrated by growth cone guidance via long-range, diffusible molecules (reviewed by Goodman, 1996; Tessier-Lavigne and Goodman, 1996). Gunderson and Barrett (1979) provided the first direct evidence that axons can recognize diffusible molecules secreted by target cells when they demonstrated that sensory neurons respond to gradients of soluble NGF in vitro. This study sparked a resurgence in the theory of chemotropism and the search for candidate diffusible growth cone guidance signals. The identification of diffusible guidance signals was aided by the development of the collagen gel assay developed by Lumsden and Davies in the early 1980s. This assay consists of coculturing, in collagen gel matrices, explants of different tissues placed at a distance. To date, many molecules from a variety of systems have been shown to guide axons from a distance in vitro (e.g. Lumsden and Davies, 1983; Tessier-Lavigne et al., 1988; Pini, 1993; Serafini et a l , 1994; Messersmith et al., 1995; Caroni, 1998). Though there are few examples of molecular gradients guiding axons in vivo, chemoattraction and chemorepulsion are widely accepted as mechanisms of axon guidance independent from short-range, contact-mediated guidance. 13 Interestingly, the distinction between short-range and long-range guidance mechanisms, attraction and repulsion is not specific to the gene family. Members of the same family of guidance molecules can be cell-surface or secreted (Culotti and Kolodkin, 1996), and even individual guidance molecules have been shown to be bifunctional, conferring repulsion or attraction depending on the growth cone (Song et al., 1998). One large family of secreted and transmembrane glycoproteins demonstrated to function within the nervous system in axon pathfinding, branching, and target selection is the Semaphorins (Mark et al., 1997). The first semaphorins identified were chick Collapsin-1, a secreted semaphorin shown to collapse D R G growth cones from a distance in vitro (Luo et al., 1993), and grasshopper Sema la, a transmembrane semaphorin shown to display both inhibitory and permissive functions in vivo (Kolodkin et al., 1992; Wong et al., 1997). In Chapter 3, we characterize a novel grasshopper secreted semaphorin, gSema 2a, with particular focus on possible roles for this molecule in pioneer growth cone pathfinding (Isbister et al., 1999). In addition, we investigate possible combined and independent roles for the secreted Sema 2a and transmembrane grasshopper semaphorin, gSema la, on growth cone pathfinding. Growth cone guidance cue detection mechanisms It has become increasingly apparent that an expansive and diverse array of both positive and negative signals guides neuronal growth cones through preexisting tissue towards their appropriate targets. These signals are constantly changing both temporally and spatially, thus, growth cones must continually combine and integrate information. Growth cone steering decisions are based on the outcome of this integration and the resultant intracellular signalling events that regulate the cytoskeleton. Remarkably, neuronal growth cones pathfind over distances that can be many times than their own diameter, demonstrating their capacity to not only integrate multiple incoming signals, but to reliably detect and amplify relatively small spatial changes in guidance information. How growth cones detect and 'read' the array of information presented by the environment is unknown. Clearly, guidance signals interact with proteins on the growth cone surface; however whether information can be encoded in the expression pattern of the signal, i.e. graded versus uniform distribution, or how the growth cone might discriminate between possible differences in expression pattern are questions awaiting further investigation. Growth cone guidance by molecular gradients The theory that molecules released from targets can guide developing neurons through preexisting tissue can be traced back as far as over a century ago to the writings of Ramon y Cajal (see review by Nieto, 1996). However it was not until the 1940s that research into chemotropism gained momentum, mostly as a result of Roger Sperry's studies on the newt visual system where he assessed the regenerative capacity of retinal axons to their targets in the tectum following severing of the optic nerve. After years of extensive studies, Sperry published his chemoaffinity hypothesis (1963) which proposed that neurons acquire unique molecular labels early in development and that topographic maps are generated by matching molecules on the pathfinding growth cone and the target. Sperry provided evidence for a high degree of specificity in the establishment of neuronal connectivity, and further proposed that growth cones can be guided by gradients of soluble signals emanating from their targets. Although additional mechanisms are now known to contribute to accurate connectivity (Goodman and Shatz, 1993), the basic tenets of Sperry's chemoaffinity hypothesis are widely accepted. One major caveat of the early versions of the chemoaffinity hypothesis was the unrealistic number of molecules that would be required to tag every neuron with an individual label. To address this concern, Sperry later revised his hypothesis and proposed that specificity 15 could be brought about by the superimposition of at least two gradients, providing each axon with a particular "latitude and longitude". In fact, this mechanism has recently been demonstrated in the retinotectal system where Eph receptors and their ligands are expressed in overlapping, complementary gradients within the retina and tectum (reviewed by Friedman and O'Leary, 1996; Braisted et al., 1997; Drescher et al., 1997). Although many in vitro studies have confirmed axon guidance by gradients (reviewed by Nieto, 1996; Goodman, 1996; Varela-Echavarria and Guthrie, 1997), there are very few examples of protein gradients in vivo and, as a consequence, few studies have investigated axon guidance by this mechanism in vivo. Our initial characterization of gSema 2a indicated this protein is expressed in a gradient during the period of Til pioneer pathfinding (Chapter 3); in Chapter 4 we further investigate this possibility. In addition, we explore whether gradient shape has functional significance for the pathfinding of growth cones in vivo. Mechanisms of gradient-reading. Although considerable effort has been directed at characterizing chemotactic molecules and their receptors, surprisingly little is known about the mechanisms that neuronal growth cones employ to detect these gradients. It is proposed that the growth cone detects chemotactic gradients by evaluating the change in concentration of guidance cue over its spatial extent, and then moves in the direction of increasing concentration for chemoattractant cues, or decreasing concentration, in the case of chemorepellent cues. Ultimately, for a growth cone to reliably pathfind up or down a gradient, a difference in ligand concentration across the growth cone must be detectable above the background noise. Two possible mechanisms for growth cone detection of small changes in external gradients have been proposed and they differ on which aspect of the change in concentration across the growth cone is most important, the absolute change or the fractional change (Walter et 16 al., 1990; Goodhill and Baier, 1998; Goodhill, 1998). Similar models are under debate in chemotaxis of cellular slime mold, leukocytes and other eukaryotic cell types (Devreotes and Zigmond, 1988; Parent and Devreotes, 1999). However, until recently, distinguishing the mechanism employed by neuronal growth cones and investigating the role of absolute concentration and gradient steepness in growth cone pathfinding, has been limited by the scarcity of functional data on gradients of neuronal guidance molecules in vivo. In Chapter 4, we use our functional Sema 2a gradient data to explore theoretical mechanisms of growth cone gradient detection in vivo. Summary The process of axonal pathfinding is classically considered activity independent, with molecules in the environment guiding growth cones towards their appropriate target. We took advantage of the ease of access and manipulation of the embryonic grasshopper Ti 1 pioneer projection to investigate mechanisms of neuronal growth cone guidance in vivo. The experiments described in this thesis provide insight into the mechanisms growth cones employ to detect and integrate the varied forms of axonal guidance information within the developing organism. Differential adhesion to the extracellular environment is one mechanism proposed to guide growth cone pathfinding. In Chapter 2 we describe an in vivo adhesion assay developed to test whether filopodial-substrate adhesion is sufficient to determine T i l growth cone steering events. We demonstrate that filopodial retraction rate following treatment with cytochalasin D or elastase reflects the degree of filopodia-substrate adhesivity. Therefore, we hypothesized that i f differential adhesion across the growth cone governs T i l pathfinding in vivo, then correctly oriented filopodial and incorrectly oriented filopodial retraction rates should differ following CD 17 or elastase treatment. However, we observed no difference between retraction rates of filopodia extending towards the correct target and filopodia extending away from the correct target. The homogeneity in filopodial retraction rates, even among turning growth cones where differential adhesivity might be expected to be greatest, strongly indicates that differential adhesion does not govern T i l pioneer neuron pathfinding. Therefore, while adhesive molecules are likely necessary for axon extension as well as for a variety of specific cell-cell interactions, our results indicate that differential adhesion is not the only force guiding neuronal growth cones towards their appropriate targets. In vitro studies have demonstrated that diffusible molecules can also influence growth cone pathfinding, indicating that mechanisms distinct from adhesion can indeed contribute to guidance. In Chapter 3 we investigate whether secreted molecules can affect neuronal growth cone pathfinding in vivo. We demonstrate that a novel secreted semaphorin in grasshopper, gSema 2a, functions as a chemorepulsive guidance molecule critical for determining both the initial direction and subsequent pathfinding events of the T i l axon projection. Our results establish that in addition to contact-mediated mechanisms, secreted molecules, such as semaphorins, can also steer growth cones. Building upon our initial characterization of gSema 2a, in Chapter 4 we demonstrate that gSema 2a is expressed in overlapping gradients within the developing limb bud during T i l growth cone pathfinding. Furthermore, we provide evidence that the steepness of the gradient, not absolute levels of protein expression, provides the critical chemorepulsive guidance information. Finally, using this functional gradient data we begin to elucidate some of the mechanisms growth cones may employ to decipher patterned expression of guidance molecules in vivo. 18 II. FILOPODIAL ADHESION DOES NOT PREDICT GROWTH CONE STEERING EVENTS IN VIVO. Introduction Growth cones of developing neurons must discriminate between a variety of environmental cues to accurately pathfind and establish connections with their targets. Filopodia extend and retract from the growth cone, actively exploring the environment and altering the direction of growth cone advance in response to these cues (reviewed by Heidemann et. al., 1990; Lin et. al., 1994; Kater and Rehder, 1995; Mitchison and Cramer, 1996; Lauffenburger and Horwitz, 1996). Although it is established that filopodia are necessary for accurate growth cone guidance (Bentley and Toroian-Raymond, 1986; Chien et. al., 1993; Zheng et al., 1996), it remains unclear how filopodia integrate and transduce guidance information from multiple external cues into motile forces. Early in vitro experiments conducted by Letourneau (1975) indicated that growth cones may select the most adhesive pathway available for migration. These experiments supported a model for growth cone steering based on differential expression of adhesive molecules in the environment. More recently, various in vitro studies have quantified the adhesion of neurites on extracts of purified embryonic substrate-bound adhesion molecules (Gundersen, 1987; Gundersen, 1988; Calof and Lander, 1991; Lamoureux et. al., 1992; Lemmon et. al., 1992; Gomez and Letourneau, 1994). The majority of these studies showed little correlation between adhesion and neuron outgrowth, indicating that endogenous substrates may not exert their effects on axon guidance principally via relative adhesiveness. Nonetheless, observation of isolated growth cones on artificially simple substrates may not reflect in vivo mechanisms of growth cone guidance. However, in vivo growth cone adhesiveness has been difficult to quantify and, therefore, the role of adhesion in axonal pathfinding in vivo could only be inferred from observation of growth cone morphology and dynamics (Caudy and Bentley, 1986a; Myers and Bastiani, 1993), or in preparations devoid of extracellular matrix (Condic and Bentley, 1989a,b). Therefore, although it is accepted that adhesion to the substrate is necessary for normal outgrowth, the role of filopodial-substrate adhesion in directing axonal pathfinding in vivo remains unclear. Axonal pathfinding has been studied extensively in the embryonic grasshopper limb where the T i l pioneer growth cones display a strong preference for migration along a stereotyped pathway (reviewed in Bentley and O'Connor, 1992; Sanchez et. al., 1995). T i l pioneer growth cone filopodia, which are necessary for accurate pathfinding, contact a variety of substrates including epithelial and neuronal cells and an extensive basal lamina (Bentley and Torian-Raymond, 1986; Anderson and Tucker, 1988; Condic and Bentley, 1989a,b). In the present study we use time-lapse imaging to demonstrate that regional cues along the Ti 1 pioneer pathway not only provide guidance information but also are capable of increasing extension rates of target-directed filopodia. To elucidate the role of substrate adhesion in these growth cone steering events, we developed an assay for quantifying substrate adhesivity for individual filopodia within a given T i l growth cone. Using this assay we examined whether there is a correlation between the direction of T i l pioneer growth cone extension and the filopodial-substrate adhesiveness. We demonstrate that in vivo growth cone steering events are not correlated with filopodial-substrate adhesion, thus indicating differential filopodial adhesion to the extracellular environment does not govern growth cone pathfinding in vivo. 21 Materials and Methods Embryo culture and neuronal labeling. Schistocerca gregaria embryos were obtained from a colony maintained at the University of British Columbia. Eggs were staged, sterilized and the T i l limb buds from 30.5%-33.5% embryos were dissected as previously described (Bentley et. al., 1979; Caudy and Bentley, 1986b). Embryos were placed ventral side down on a poly-L-lysine coated cover slip (6 mg/ml), the dorsal epithelium of the T i l limb buds was cut lengthwise, and unrolled flat to expose the pioneer pathway. A suction pipette was used to remove the mesodermal cells overlaying the limb epithelium, exposing the T i l neurons. Limb preparations were bathed in a modified RPMI culture medium and viewed with Nomarski optics on a Nikon inverted compound microscope (O'Connor et. al., 1990). T i l neurons were labeled with D i l (membrane marker; Molecular Probes) by gently touching to the cell body a micropipette previously coated with D i l and air dried. Rhodamine-phalloidin (F-actin marker; Molecular Probes) injections were carried out as previously described (O'Connor and Bentley, 1993), and injected neurons were double labeled with DiO (membrane marker; Molecular Probes). Time-lapse microscopy. Labeled T i l neurons were illuminated with a Nikon 100 W halogen light and the appropriate filter set (Chroma Tech), and shuttered with a computer-controlled Lambda 10-2 shutter (Sutter Instrument Co.). Neurons were imaged with a Princeton Instruments MicroMax CCD camera (Kodak chip K A F 1400) and digitized with Princeton Instruments Winview 1.6.2., except Figure 3 which was imaged with a Photometries camera (Kodak chip K A F 1400) with Perceptics Software. Elapsed time was automatically recorded for each image. 22 Three to five labeled T i l pioneer neurons were imaged until one growth cone was selected for full analysis. Growth cones displaying a complex array of filopodia required two to three focal planes imaged per time point and the parts in focus were combined into a digital montage. To record in vivo growth cone behaviour, labeled T i l neurons were imaged for 1 - 3 hours pre cytochalasin D (CD) or elastase application. This time frame ensured only healthy, normally advancing growth cones were selected for full analysis. A selected neuron was then imaged exclusively for 1- 3 hours after the addition of drug. Healthy labeled T i l neurons in the dish not selected for full analysis were also imaged at the last time point before drug application, the first post drug time point, and the final time point of the experiment. After image collection, preparations were fixed, and the T i l neurons and guidepost cells labeled with neuron-specific antibodies to confirm their positions (Jan and Jan, 1982). Cytochalasin D and Elastase treatments. Cytochalasin D (2uM) or elastase (0.03%) was introduced to limb preparations by replacing the original media in the dish with culture media containing the drug or enzyme (MacLean-Fletcher and Pollard, 1980; Bentley and Toroian-Raymond, 1986; Forscher and Smith, 1988; Condic and Bentley, 1989a,b), and image collection then continued for 1 - 3 hours post treatment. In a few cases, growth cones were imaged for up to 12 hours post media exchange. The time lapse between the last image pre treatment and the first image post treatment was usually within 5 minutes, and in many cases as quickly as 1 minute. Determining on-axis and off-axis fdopodia. Growth cones were imaged at various developmental stages of the T i l pioneer pathway and categorized as either migrating within the intrasegmental femur epithelium or migrating within the trochanter segmental epithelium (Fig. 1). These growth cones were further 23 categorized as either non-turning growth cones or growth cones in the process of turning. There are two abrupt stereotyped turning events in the T i l pioneer pathway. The first is at the Tr cell, located within the trochanter segmental epithelium, where growth cones turn ventrally to migrate down the trochanter epithelium. The second is where growth cones turn proximally towards the C x i cells and migrate away from the trochanter segmental epithelium (Fig. 1). Each filopodium was categorized as on-axis or off-axis. On-axis filopodia were those extending along the axis of the stereotyped T i l pioneer neuron migration trajectory (correct orientation); off-axis filopodia were those extending off the stereotyped migration axis by an angle greater than 45 degrees (incorrect orientation; Fig. 2A). For growth cones in the process of turning ventrally at the Tr cell, on-axis and off-axis filopodia were often distinctly distributed into ventral and dorsal populations respectively (Fig. 2B). Similarly, on- and off-axis filopodia of growth cones in the process of turning proximally towards the C x i cells were clearly distributed into proximal and distal populations respectively (Fig. 2C). The location of the filopodial tip was the criterion for determining on-axis versus off-axis location, few filopodia had their base and tip in different categories (7 of 359). Filopodial measurement and analysis. Filopodial length was measured for each time point using NIH Image. Growth cones that required multiple focal planes were digitally montaged in order to accurately measure the entire length of individual filopodia. Measurements were taken from the centre of the growth cone, or branch, to the filopodial tip. Measuring from the centre eliminated error from misinterpreting growth cone, or branch, diameter changes as filopodial length changes. Filopodia that retracted completely into the growth cone before addition of CD or elastase were not included. The results are based on the behaviour of 359 filopodia from 22 growth cones for which we have complete data sets before and after media exchange. 24 Figure 1. Schematic of Til pioneer neuron pathway. The pair of sibling T i l pioneer neurons arises from the underlying epithelium between 29-30% of embryonic development. At approximately 30.5% of development the T i l growth cones emerge from their cell bodies and begin to extend axons proximally along the limb axis towards the CNS. The T i l growth cones may or may not contact the Fe guidepost cell on route to the Tr cell. Once contact with the Tr cell has been made, usually around 33% of development, the T i l growth cones reorient circumferentially and extend ventrally along the trochanter epithelium. A second turning event in the T i l pioneer pathway occurs at about 34% development and is typified by a distinct reorientation toward the C x i guidepost cells. By 35% the T i l pioneer growth cones have extended proximally from the C x i cells into the CNS. Dashed lines indicate limb segment boundaries. 25 Femur 26 Figure 2. Categorization of growth cone filopodia as on-axis or off-axis. A, A representative non-turning T i l growth cone imaged during migration towards the Tr cell. On-axis filopodia are defined as those extending along the proximal/distal limb axis, off-axis filopodia are those extending off the stereotyped migration axis by an angle greater than 45 degrees (6). B, A T i l growth cone imaged in the process of turning at the Tr cell (*). Off-axis filopodia are those filopodia that are extended dorsally in the incorrect direction while on-axis filopodia are defined as those filopodia extended in the correct ventral direction. This distribution of filopodia should maximize the chance of observing any adhesive differences between the ventral and dorsal epithelium. C, A T i l growth cone reorienting towards the C x i cells. Similar to the abrupt turn at the Tr cell, on- and off-axis filopodia are distinctly distributed into proximal and distal populations, respectively. Scale bar, 10pm. 27 28 Previous analysis of T i l filopodial behaviour has determined that individual filopodia extend at relatively slow rates (on average approximately 0.67pm/min) (O'Connor et al. 1990). This allowed us to sample filopodial lengths at relatively long time intervals (10-15 minutes), thus reducing the amount of exposure to the U V light source. However, to ensure that these time intervals were sufficient to accurately reflect filopodial behaviour, we imaged some growth cones at higher frequencies. Also, because we could never be positive that filopodia did not extend and retract multiple times between acquired images, we calculated the net change in length over a period of 60 minutes before and after the addition of CD or elastase. Rates of extension and retraction over the 60 minutes before and after addition of drug were compared between on-axis and off-axis filopodial populations. For analysis of the difference in retraction rates between filopodial populations we also compared length change during the first time period following drug application. A two-tailed unpaired t-test was used to compare the average retraction rate of all filopodia (both on-axis and off-axis) from growth cones within the femur intrasegmental epithelium to the average retraction rate of all filopodia from growth cones within the trochanter segment boundary epithelium. For the remaining experiments, two-tailed unpaired t-tests were performed to compare on- and off-axis filopodia for individual growth cones. To pool data from all growth cones, change in filopodial length was standardized (converted to z-scores), and two-tailed unpaired t-tests were performed on the pooled standardized data. 29 Results Cytochalasin D disrupts the internal cytoarchitecture of Til filopodia. Cytochalasins are fungal metabolites that inhibit F-actin elongation by capping actin filament ends in a reversible, dose-dependent manner (MacLean-Fletcher and Pollard, 1980; Schliwa, 1982; Cooper, 1987; Sampayh and Pollard, 1991). Although inhibition of actin polymerization with cytochalasins blocks filopodia extension and motility (Marsh and Letourneau, 1984; Bentley and Toroian-Raymond, 1986), retrograde F-actin flow continues (Forscher and Smith, 1988). Previous in vitro studies have demonstrated that this continued F-actin retrograde flow, in the absence of polymerization, results in F-actin clearance from the growth cone lamellipodia and filopodia within 1 -3 minutes, and usually after 2 - 4 minutes the filopodia have started to retract into the growth cone (Forscher and Smith, 1988). Using rhodamine-phalloidin (Rh-phalloidin), we monitored actin dynamics in T i l filopodia following treatment with cytochalasin D. To confirm that C D disrupted filamentous actin (F-actin) in T i l growth cone filopodia, selected T i l cell bodies were injected with Rh-phalloidin and the membrane was labeled with DiO. Images of F-actin dynamics and the growth cone morphology were collected before and after treatment with CD (Fig. 3). Immediately following CD addition, rhodamine-phalloidin labeled actin began to disassemble and by 5-10 minutes was reduced to punctate staining within the filopodia. By 30 minutes Rh-phalloidin fluorescence was restricted to the growth cone body and large branches (Fig. 3D). Although CD rapidly removed the F-actin within the T i l filopodia, the majority of the filopodia remained extended (Fig. 3C). We attribute the slow retraction of T i l growth cone filopodia to the gradual loss of filopodial membrane adhesive contacts with the extracellular environment. In comparison to CD treated growth cones in vitro, the T i l filopodia retract relatively slowly, possibly reflecting the complexity of the extracellular environment surrounding the filopodia. Filopodial 30 Figure 3. Cytochalasin D disrupts internal cytoarchitecture of Til filopodia. Images of a T i l neuron were collected as the growth cone migrated ventrally along the trochanter epithelium. A-D: Individual pioneer neurons were labeled with DiO to label the cell membrane (A) and rhodamine-phalloidin was intracellularly injected to label the F-actin (B). Rh-phalloidin staining can be seen within the growth cone body and along the length of the filopodia (B, arrowheads). C, DiO image of the same growth cone as in (A) 30 minutes following CD application. Many of the filopodia remain extended (C, arrowheads) despite the disruption of the actin cytoarchitecture with cytochalasin D. D, Rh-phalloidin image of the same growth cone 30 minutes following CD treatment. Rh-phalloidin staining was undetectable in filopodia (D, arrowheads) and reduced to punctate staining within the growth cone body. Scale bar, 10pm. 31 B Rh-PrClloidin preCD DRh-Phalloidin postCD 32 retraction began 2-3 minutes after addition of CD, thus suggesting that the mechanisms involved in generating filopodial retraction occur over the order of minutes. For many filopodia, retraction occurs over a number of hours (see Figs 6, 7), with eventual complete retraction of the majority of filopodia (Fig. 4). For example, figure 4 shows the distribution of filopodia extending from a growth cone that is migrating down the trochanter epithelium. After 12 hours of exposure to CD the growth cone had extended approximately 15 pm, however only a few large filopodial branches remained. These branches were evenly dispersed around the growth cone with two on-axis and two off-axis branches remaining. Filopodial retraction rate following cytochalasin D or elastase application reflects filopodial:substrate adhesion. Previous observations of growth cone morphology and direct tests of growth cone adhesion have illustrated that grasshopper trochanter segment epithelium is more adhesive than femur intrasegmental epithelium (Caudy and Bentley, 1986a,b; Condic and Bentley, 1989a,b). In order to test the validity of our in vivo adhesion assay we examined whether filopodial retraction rates following the addition of CD or elastase reflect these known differential adhesivities. We predicted that filopodia in contact with the more adhesive trochanter segment epithelium would retract slower following the removal of F-actin or the basal lamina adhesive interactions. To test this we compared the average filopodial retraction rate of growth cones migrating in the more adhesive trochanter epithelium with the average filopodial retraction rate of growth cones migrating in the less adhesive femur epithelium. Figure 5 shows the average of both on- and off-axis filopodial retraction over the 60 minutes following CD or elastase application. For the CD trials, analysis of 60 filopodia from 4 growth cones migrating in the less adhesive femur epithelium revealed an average retraction of 10 ± 0.6pm during the 60 minutes following C D application. In contrast, analysis of 87 33 Figure 4. The majority of filopodia eventually retract after cytochalasin D treatment. Images of a D i l labeled T i l neuron migrating along the trochanter epithelium. A , 1 minute following CD application filopodia have not yet retracted into the growth cone. B, 12 hours following C D application most of the filopodia have retracted into the growth cone. The growth cone has extended approximately 15pm. Scale bar, 10pm. 34 A 1 min postCD B 12 hours postCD 35 Figure 5. Filopodial retraction rate following cytochalasin D or elastase application accurately reflects in vivo substrate adhesion. Average filopodial retraction during 60 minutes post CD application was determined for 4 growth cones migrating along the low affinity (less adhesive) femur epithelium (n = 60 filopodia), and for 5 growth cones within the high affinity (more adhesive) trochanter epithelium (n = 87 filopodia). Similarly, average filopodial retraction during 60 minutes post elastase application was determined for 3 growth cones migrating along the femur epithelium (n = 66 filopodia), and for 4 growth cones within the trochanter epithelium (n = 69 filopodia). Average retraction rate was determined using both on-axis and off-axis filopodia. Following either CD or elastase application, the average filopodial retraction rate for growth cones migrating along the less adhesive femur epithelium was significantly greater than the retraction rate for filopodia of growth cones interacting with the more adhesive trochanter segment epithelium (* p < 0.001, ** p < 10"5 ). Error bars: ±SEM. 36 35 j 30-^=1 c o m mmm 25---4—• o i_ 20-0 C£ 15-"crj "D O 10-Q_ O 5 -L L 0--I I Femur epithelium • • Trochanter epithelium i 1 CD Elastase filopodia from 5 growth cones migrating within the more adhesive trochanter epithelium exhibited a significantly slower average retraction of 7 ± 0.8pm during the 60 minutes following CD application (t test, p < 0.001). In addition, analysis of the averaged filopodial length change during the first time period after CD addition revealed a significantly slower retraction rate of filopodia located in the trochanter epithelium (t test, p < .0005). Thus, slower filopodial retraction rates following CD application of growth cones migrating within trochanter segment epithelium accurately reflect filopodial contact with the more adhesive substrate. Previous experiments have demonstrated that elastase disrupts growth cone-basal lamina adhesive contacts (Condic and Bentley, 1989a,b), thus leaving only the actin cytoskeleton and epithelial adhesive contacts to oppose filopodial collapse. Therefore, we confirmed the CD results by measuring T i l filopodial retraction rates following the removal of basal lamina adhesive interactions with elastase (Fig. 5). We analyzed 4 growth cones within the trochanter epithelium and 3 growth cones within the femur intrasegmental epithelium. Consistent with the CD results, we found growth cones interacting with the more adhesive trochanter epithelium displayed slower filopodia retraction rates in comparison to filopodia extending along the less adhesive femur epithelium (r test, p < 10"5). These results support the utility of filopodial retraction rate as an assay of filopodial-substrate adhesivity during growth cone steering events. Filopodial behaviour of non-turning Til pioneer neurons. To record in vivo pathfinding behaviours of non-turning T i l pioneer neurons, growth cones were imaged for 1 to 3 hours following D i l labeling. Figure 6 is a representative example of the analysis for each non-turning growth cone in this study. In this example the growth cone was imaged as it migrated along the mid-femur epithelium towards the Tr cell (see Fig. 1). After the addition of CD, filopodia started to retract into the growth cone (6A, arrowheads). Analysis of individual filopodia length versus time illustrates that the majority of filopodia are dynamic, 38 Figure 6. Pathfinding behaviour of non-turning Til growth cones before and after treatment with cytochalasin D. A , Three representative images of a D i l labeled non-turning T i l growth cone migrating towards the Tr cell along the proximal-distal axis of the limb. (Left) Before addition of CD. (Center) 2 minutes after CD treatment. (Right) 30 minutes post CD treatment. Arrowheads demarcate filopodia before and after addition of CD, illustrating filopodial retraction. B, Filopodial length (y-axis) versus time (x-axis) for individual filopodia. Each trace indicates an individual filopodia. The time of CD treatment is indicated by a vertical line. (Left panel) On-axis filopodia. (Right panel) Off-axis filopodia. C, Average change in filopodial length from the last time point (y-axis) versus time (x-axis). CD treatment is indicated by a vertical line. Both on-axis and off-axis filopodia show immediate retraction after CD application. D, average change in filopodial length for 60 minutes before and 60 minutes following CD. No significant differences between on-axis and off-axis rates were observed before or after CD treatment. Error bars: ±SEM. 39 On-Axis n n Off-Axis Time (min) exhibiting periods of growth and retraction before CD treatment (Fig. 6B). On average, during the period of observation, length increased for both on-axis and off-axis filopodia in this particular growth cone and no significant difference in the growth rate of on-axis and off-axis filopodia was observed 60 minutes prior to CD (Fig. 6D; t test, p = 0.90). In the CD trials, we analyzed 11 non-turning growth cones (n = 146 filopodia); in 9 of 11 growth cones, there was no significant difference between on-axis and off-axis filopodia extension during the 60 minutes prior to the addition of CD. In 1/11 growth cones a significantly greater extension rate was observed in the on-axis filopodia during this period, while in 1/11 growth cones, a significantly greater extension rate was recorded for the off-axis filopodia. Thus, in the majority of non-turning growth cones, there was no significant difference in filopodial length changes observed between on-axis and off-axis filopodia before CD treatment. In addition, equal numbers of filopodia were distributed between on-axis and off-axis. In order to pool the data from all non-turning growth cones, the change in length for each filopodia over the 60 minutes prior to CD application was standardized by conversion to z-scores. Analysis of the pooled, standardized filopodia data confirmed there was no significant difference between on- and off-axis filopodial growth (Fig. 8^ 4; t test, p = 0.42; page 47). The frequency histogram of the on-axis and off-axis pre-CD z-scores were identically distributed, with filopodial length changes evenly distributed about their respective mean (graph not shown). We separated the non-turning growth cones into sub-groups based on the developmental stage of the T i l pathway, for example, before the Tr cell or along the trochanter epithelium. Analysis of these sub-groups also revealed no significant difference in extension rates between on-axis and off-axis filopodia (before Tr cell; t test, p = 0.41; trochanter epithelium; t test, p = 0.83). Likewise, analysis of the three non-turning growth cones from the elastase trials revealed no significant difference in filopodial extension rates (Fig. 9C; page 49). These results suggest that 41 in non-turning growth cones the dynamic behaviour of on-axis and off-axis filopodia are not significantly different. Filopodial adhesion does not predict growth cone pathfinding of non-turning Til neurons To determine the role of adhesion in T i l growth cone pathfinding in vivo, we compared the retraction rates of on-axis and off-axis filopodia after treatment with CD or elastase. We proposed that i f adhesion of filopodia to the extracellular environment predicted growth cone migration, then on-axis filopodia should retract at a reduced rate when compared to off-axis filopodia. A representative example of the analysis of a non-turning growth cone from the CD trials is shown in figure 6. A l l filopodia ceased extending and a notable retraction was observed in both on-axis and off-axis filopodia (Fig. 6B, C). To determine if there was a significant difference between on-axis and off-axis retraction rates, the average change in filopodia length was calculated for the 60 minutes following CD application for both on-axis and off-axis filopodia. No significant difference was observed (Fig. 6D; t test, p = 0.89). In addition, analysis of the averaged filopodia length change during the first time period after CD addition revealed no significant difference in retraction rates (t test, p = 0.15). For the CD trials, a total of 146 filopodia from 11 non-turning growth cones were analyzed prior to and following addition of CD. In 11 of 11 non-turning T i l neurons, analysis revealed no significant difference in retraction rates between on-axis and off-axis filopodia. Analysis of pooled, standardized filopodia also revealed no significant difference in filopodial retraction rates (Fig. SB; t test, p = 0.65; page 47). Furthermore, there was no significant difference in retraction rates even during the first time period post CD (t test, p = 0.40) or when non-turning growth cones were grouped into developmental stages (before Tr cell; t test, p = 0.97; ventral migration in trochanter epithelium; t test, p = 0.67). Representation of the post-CD z-scores of non-turning growth cones as a frequency histogram revealed a shift in all on-axis 42 and off-axis filopodial length changes to negative z-scores (graph not shown), confirming all filopodia began to retract following CD treatment. Analysis of the 3 elastase treated non-turning growth cones also revealed no difference between retraction rates of on- and off-axis filopodia (Fig. 9C; page 49). These data suggest that differential filopodial adhesion to the extracellular environment is not a governing factor in non-turning T i l neuron pathfinding in vivo. Filopodial behaviour of Til pioneer neurons during growth cone turning events The growth cones of T i l neurons that were in the process of turning were imaged for 1 to 3 hours before and following CD or elastase application. The data collection and analysis was identical to that of non-turning T i l neurons described above. Figure 7 is a representative example of the analysis of a turning growth cone from the CD trials. In this example a T i l growth cone turned proximally towards the C x i cells (Fig. 1A). As the main on-axis branch extending towards the C x i cells enlarged (open arrow), the large off-axis branch progressively collapsed (filled arrow). This shifting of the growth cone mass towards the main on-axis branch was accompanied by extension of on-axis filopodia (Fig. 7A, arrowheads). Immediately following addition of CD, what remained of the large off-axis branch continued to collapse (Fig. 1A, solid arrow) and the off-axis and on-axis filopodia began to retract (Fig. 1A right panel, arrowheads). However, the main on-axis branch remained enlarged following CD application (Fig. 1A right panel, open arrow). The persistence of the main on-axis branch after disruption of the actin cytoarchitecture most likely indicates that microtubules have invaded the main on-axis branch to form the emerging nascent axon (Sabry et. al., 1991). As observed with non-turning growth cones, filopodial number was evenly distributed on- and off-axis and the majority of filopodia exhibited periods of growth and retraction before CD application (Fig. IB). Surprisingly, when changes in filopodial length were calculated for each time interval and averaged for both on and off-axis filopodia, a striking disparity in 43 Figure 7. Pathfinding behaviour of a Til growth cone turning toward the Cxi cells before and after treatment with cytochalasin D. A , Four representative images of a D i l labeled T i l growth cone turning towards the C x i cells. The first three images are before addition of cytochalasin D, the rightmost image is 5 minutes following CD treatment. Arrowheads demarcate filopodial extension before and retraction after addition of cytochalasin D. {Open arrow) Main on-axis branch; {solid arrow) Main off-axis branch. Following addition of cytochalasin D, the large off-axis branch has narrowed markedly {solid arrow); whereas, the principal on-axis branch remains enlarged, possibly indicating microtubule invasion and nascent axon formation {open arrow). B, Filopodial length (y-axis) versus time (x-axis) for individual filopodia. The time of CD treatment is indicated by the vertical line. (Left panel) On-axis filopodia. (Right panel) Off-axis filopodia. C, Average change from the last time point in filopodial length (y-axis) versus time (x-axis). The time of CD application is indicated by the vertical line. D, Average change in filopodial length for 60 minutes before and 60 minutes following CD. A significant difference between on-axis and off-axis filopodial growth was observed before addition of CD (* p < 0.05). No significant difference was observed between on-axis and off-axis filopodial retraction following addition of CD. Error bars: ±SEM. 44 B 80 E 604 O) c 0 40] CO §.20] o • 40 min pre CD A ^ • 5 min pre CD On-Axis 80 - I E a. 60 J m .§40 TJ §.20 o o Off-Axis CD *• 3. * * *• * : A s »^ -»— • 0 50 100 150 200 250 Time (min) 0 50 100 150 200 250 Time (min) c 6-i E 4-2-(D CD o-C CO -2 O -4-D) -6-C <D -8-_ l •10J D 1 CD -a- On-Axis — Off-Axis On-Axis Off-Axis 0 50 100 150 200 250 Time (min) 45 filopodia extension rates was observed between on-axis and off-axis filopodia during the T i l turning event. On-axis filopodia showed greater extension during the 60 minutes before CD application than off-axis filopodia (Fig. IC, D; t test, p = 0.03). This disparity occurred in all of the turning growth cones examined. Analysis of the pooled z-scores from a total of 78 filopodia from 4 growth cones revealed that this difference in on-axis versus off-axis extension during the 60 minutes pre CD was consistent and highly significant only at turning points in the T i l pioneer pathway (Fig. SA; t test, p < 10"7). The frequency distribution of the pooled turning z-scores revealed both on-axis and off-axis z-scores were evenly distributed about their respective means, with the on-axis filopodia mean 1.3 standard deviations greater than off-axis filopodia (graph not shown). This separation in the normally distributed on-axis and off-axis z-scores indicated that the significant difference between extension rates was not the result of outliers (also confirmed by the height of the error bars in Fig. 8). We analyzed the pooled raw data to determine whether the statistical difference observed between on-axis and off-axis filopodial extension before CD in turning growth cones was a result of increased growth in the on-axis filopodia or a relative decrease in off-axis filopodial extension. This analysis revealed that the average on-axis filopodia extension rate was over 300% greater than the average off-axis filopodia extension rate (Fig. 85; t test, p < 10"5). Interestingly, this increase in on-axis extension in turning growth cones was also significantly greater (over 100%) than on-axis filopodial extension in non-turning growth cones (Fig. SB; t test, p = 0.005). There was no significant difference in the off-axis filopodial extension rates of turning and non-turning growth cones (Fig. SB; t test, p = 0.11). Furthermore, analysis of the four turning growth cones in the elastase trials also revealed these trends (Fig. 9; n = 69 filopodia), increasing the number of turning growth cones to 8. These results indicate that in vivo T i l pioneer neuron turning events are characterized by an increased on-axis filopodial extension rate during turning events. 46 Figure 8. In vivo Til growth cone pathfinding behaviours and filopodial extension rate are not governed by differential filopodial substrate adhesion. A , The average standardized length change (z-score) 60 minutes before and 60 minutes following CD application for 11 non-turning and 4 turning growth cones (n = 224 filopodia). There was no significant difference between on-axis and off-axis filopodial growth in non-turning growth cones during the 60 minutes before CD application. However, a highly significant difference between on-axis and off-axis growth 60 minutes prior to CD application was observed in turning growth cones (n = 78 filopodia; ** p < 10"7). There was no significant difference between on-axis and off-axis filopodial length change observed in non-turning or turning growth cones following CD application. B, Average length change for 60 minutes prior to CD application and 60 minutes post CD application calculated using non-standardized raw data (similarity in frequency distributions determined by Kolgormov-Smirnov tests). Statistical difference observed between on-axis and off-axis filopodial extension in turning growth cones was a result of increased growth in the on-axis filopodia versus a relative decrease in off-axis filopodial extension (** p < 10"5). On-axis filopodial extension during growth cone turning events is significantly greater than on-axis extension during non-turning events (* p < 0.01). No significant difference was observed in the raw data between on-axis and off-axis filopodial retraction following addition of CD for either non-turning or turning growth cones. Error bars: ±SEM. 47 A Pooled Z-Scores Non-Turning CO CD i — O o CO I N 1 T 0.5 0 & -0-5 + 5 - i t 6 -1-5t £ -2 c -2 5 •H- CD lOn-Axis lOff-Axis Turning • On-Axis • Off-Axis B Pooled Raw Data ' P * A Non-Turning t20n-Axis •Off-Axis Turning • On-Axis • Off-Axis 48 Figure 9. Elastase experiments confirm that in vivo filopodial extension rate and pathfinding behaviours of Til growth cones are not governed by differential substrate adhesion. A , Three representative images of a pair of sibling D i l labeled T i l growth cones in the process of turning ventrally within the trochanter; analysis presented in (B) is of the top growth cone. The first image is before addition of elastase, the second two images are following elastase treatment. Arrowheads show filopodial retraction after addition of elastase. B, Average change in filopodial length for 60 minutes before and 60 minutes following elastase. A significant difference between on-axis and off-axis filopodial growth was observed before addition of elastase (* p < 0.005). No significant difference was observed between on-axis and off-axis filopodial retraction following addition of elastase. C, Pooled elastase data from 7 growth cones (135 filopodia) illustrating average length change for 60 minutes prior to elastase application and 60 minutes post elastase application (calculated using non-standardized raw data, similarity in frequency distributions determined by Kolgormov-Smirnov tests). The on-axis filopodia of turning growth cones extend at an increased rate (*** p < 10"6, * p < 0.05); however, no significant difference was observed between the on-axis and off-axis retraction rates following elastase for either turning or non-turning growth cones. In the elastase trials, all non-turning growth cones were migrating in the femur and all turning growth cones were at the Tr cell within the trochanter segment epithelium. Therefore, the pooled raw data also reveals the significantly slower filopodial retraction of growth cones interacting with the more adhesive trochanter epithelium (** p < 10"5). Error bars: ±SEM. 49 C Pooled Raw Data *** ** 5 0 Filopodial adhesion does not predict Til growth cone turning events Although T i l growth cones exhibit a number of different morphologies at decision points (O'Connor et al., 1990), we found that filopodia were evenly distributed between on- and off-axis sectors, thus suggesting that asymmetric distribution of filopodia does not predict growth cone steering events. Considering filopodial-substrate adhesion is greater in the regions where T i l growth cones commit to turning decisions, for example at the Tr cell in the trochanter epithelium, we hypothesized that a differential adhesion gradient across the growth cone may underlie the growth cone turning event and observed increase in filopodial extension. Therefore, to determine the role of filopodial adhesivity in T i l growth cone turning, we compared the retraction rates of on-axis and off-axis filopodia after treatment with CD or elastase. Figure 7 illustrates a representative example of the analysis of a turning growth cone from the CD trials. Consistent with non-turning growth cones, filopodia ceased extending and began retracting into the growth cone following CD application (Fig. IB, Q . To determine i f there was a significant difference between on-axis and off-axis retraction rates, the average change in filopodial length was calculated for the 60 minutes following C D application. No significant difference was observed (Fig. 7D; t test, p = 0.33), thus suggesting the greater net extension of on-axis filopodia was not due to greater filopodia-substrate adhesiveness. In addition, analysis of the averaged filopodial length change during the first time period after CD addition confirmed there was no significant difference in filopodial retraction rates (r test, p = 0.14). We analyzed 4 turning growth cones in the CD trials (n = 78 filopodia); in all T i l growth cones examined there was no significant difference in retraction rates between on-axis filopodia and off-axis filopodia. Analysis of the pooled z-scores confirmed there was no significant difference in retraction rates during the 60 minutes following CD application (Fig. %A; t test, p = 0.40) or during the first time period post CD (t test, p = 0.96). In addition, similar to non-51 turning growth cones, the frequency histogram demonstrated a consistent shift in all filopodial length changes to negative z-scores (graph not shown). Furthermore, we analyzed 4 turning growth cones in the elastase trials (n = 69 filopodia); again consistent with the CD results, there was no significant difference between on- and off-axis retraction rates (Fig. 9). These results indicate that T i l growth cone steering events, and the corresponding increase in on-axis filopodial migration rate during these turning events, are not directed by differential filopodial-substrate adhesion. 52 Discussion Using the well-characterized T i l pioneer neuron pathway of the embryonic grasshopper limb we developed an assay to test whether differences in filopodial adhesion direct growth cone steering events in vivo. We found that while filopodia in general exhibit robust adhesive interactions with the surrounding extracellular environment, there is no evidence that differential filopodial adhesion directs T i l growth cone steering events in vivo. In addition, we found that in vivo regional cues not only provide filopodia with essential guidance information but are also capable of modulating filopodial extension rate. Retraction rates of filopodia lacking F-actin or basal lamina interactions as a measure of filopodial .substrate adhesivity Traditionally, in vivo growth cone-substrate adhesion has been difficult to address due to the complexity of the in vivo environment and the impediments to access and manipulation. Thus, the role of substrate adhesion in growth cone pathfinding has remained unclear. In the present study, we took advantage of the accessibility of the well-characterized embryonic grasshopper limb T i l pioneer neuron projection to develop an assay for quantifying in vivo filopodial-substrate adhesivity. Our assay uses filopodial retraction rate, following the disruption of either the actin cytoarchitecture with cytochalasin D (CD) or the basal lamina adhesive interactions with elastase, as a measure of substrate adhesivity. To confirm the validity of our assay, we compared the filopodial retraction rates of growth cones migrating along substrates of known differing adhesivity. Evidence from observations of T i l growth cone morphology and direct tests of growth cone adhesion suggests that epithelium in the trochanter limb segment are more adhesive than intrasegmental femur epithelium (Caudy and Bentley, 1986a,b; Condic and Bentley, 1989a,b). Using our in vivo adhesion assay, we demonstrate that filopodia of T i l 53 growth cones migrating within the more adhesive trochanter epithelium retract significantly slower than filopodia of growth cones migrating within the less adhesive femur epithelium. Thus, our assay of filopodial retraction rate following application of C D or elastase accurately reflects in vivo filopodial-substrate adhesion. Our assay is based on the premise that the combined action of the actin cytoskeleton and adhesion opposes tension within filopodia, preventing them from collapsing into the body of the growth cone. With the loss of the actin cytoskeleton following CD treatment, the adhesive contacts are not sufficient to oppose tension within filopodia and therefore filopodia retract into the growth cone at a rate dependent of the degree of filopodial-substrate adhesivity. Likewise, following removal of the basal lamina adhesive interactions with elastase, the actin cytoskeleton and underlying epithelial adhesive contacts are insufficient to maintain filopodial extension. Although the integrity of F-actin is important for force generation during filopodial extension, several lines of evidence indicate a network of F-actin is not necessary for T i l filopodial-substrate adhesion. First, it has been well established in many systems, including the grasshopper T i l pioneer pathway, that an intact actin cytoarchitecture is not required for growth cone adhesion (Marsh and Letourneau, 1984; Letourneau et al., 1987; Bentley and Toroian-Raymond, 1986; Forscher and Smith, 1988; Chien et. al., 1993; Zheng et. al., 1996). In fact, in the absence of detectable F-actin, growth cones have been shown to remain adhered to a substrate and are even capable of extension (Marsh and Letourneau, 1984; Bentley and Toroian-Raymond, 1986; Chien et. al., 1993; Zheng et. al., 1996). In addition, filopodial and lamellipodial retraction often lags behind the loss of F-actin. For example, numerous Aplysia growth cone filopodia remain extended after the addition of cytochalasins, in some cases for up to 30 minutes (see Fig. ID, in Forscher and Smith 1988). Second, a variety of classical adhesive receptor complexes are known to exhibit adhesive interactions that are not associated with the actin cytoskeleton (Regen and Horwitz, 1992; Schmidt et. al., 1993; Hortschet. al., 1995; 54 Shapiro et. al., 1995; Dubreuil et. al., 1996; Kreft et. al., 1997). Third, while we cannot comment on the molecular nature of T i l filopodial-substrate adhesion, the maintenance of filopodial extension following CD clearly demonstrates continued filopodial adhesion to the extracellular environment in the absence of the actin cytoarchitecture. Differential adhesion as a model of neuronal pathfinding The T i l growth cone filopodia interact with a variety of substrates including the extracellular matrix, epithelial cells, and several pre-axonogenesis neurons. These substrates express a variety of substrate-bound guidance molecules, some of which have been shown to be necessary for accurate T i l growth cone pathfinding (Bentley and Caudy, 1983; Caudy and Bentley, 1986a; Kolodkin et. al., 1992;, Sanchez et. al., 1995; Wong et. al., 1997; Isbister et a l , 1999). How these substrate-bound adhesion molecules interact with T i l filopodia to direct growth cone steering events in vivo is not well understood. One mechanism that may direct growth cone motility is differential filopodial-substrate adhesion. Increased filopodial-substrate adhesion may be a consequence of a greater number of substrate-bound molecules binding to filopodial receptors, leading to an increased receptor-coupling to the actin cytoskeleton and a slowing of the retrograde F-actin flow. Growth cone advance, therefore, could result from attenuation of the retrograde F-actin flow combined with continued actin polymerization at the leading edge (Lin and Forscher, 1995). Alternatively, an increased receptor-coupling to the actin cytoskeleton could manifest itself intracellularly as increased tension within filopodia, creating more traction force to "pull" the growth cone forward (Heidemann et. al., 1990). Regardless of the cytomechanics, a differential expression of adhesive molecules in the environment, their respective receptors on filopodia, or alterations in the ligand-receptor binding affinity could reorient the growth cone and alter pathfinding. 55 Since it was the focus of the present study to determine whether adhesion alone is sufficient to determine growth cone steering events in vivo, we used our in vivo adhesion assay to test whether an increased substrate adhesivity could predict T i l growth cone pathfinding behaviours. If differential adhesion across the growth cone governs T i l pathfinding in vivo, then correctly oriented filopodial (on-axis) and incorrectly oriented (off-axis) filopodial retraction rates should differ following CD or elastase treatment. We found that filopodial retraction rates did not differ between on-axis and off-axis filopodia at any of the positions measured along the pathway, including during the committed turning events. The homogeneity in filopodial retraction rates, even among turning growth cones where we would predict that differential adhesion should be greatest, strongly indicates that differential adhesion does not determine T i l pioneer neuron steering in vivo. The complexity and heterogeneity among in vivo guidance mechanisms, however, cautions against prematurely concluding that differential substratum adhesivity is not involved in any growth cone guidance situation. For example, although our results establish that spatial differences in filopodia-substrate adhesion are not sufficient to determine T i l pioneer growth cone pathfinding events, there may exist in vivo situations where spatial heterogeneity in substratum adhesion across the body of a growth cone is sufficient to regulate steering events. In this study filopodial and growth cone behaviours were also examined for at least one hour prior to the addition of CD or elastase. This provided us with information about in vivo growth cone pathfinding behaviours at various decision points along the T i l pioneer pathway. We observed no consistent disparity between on-axis and off-axis extension rates in the non-turning growth cones. To our surprise, however, we observed a 3-fold increase in on-axis filopodial extension rate during T i l growth cone turning events. Furthermore, when the on-axis filopodia extension rate for turning growth cones was compared to the extension rate for on-axis filopodia of non-turning growth cones the difference was still significant; the turning filopodia 56 extended nearly twice the distance during the 60 minutes pre drug. Interestingly, a previous in vitro study demonstrated that model guideposts, composed of laminin-coated beads, not only provide directional guidance information to dissociated dorsal root ganglion neurons, but also cause a sustained 2.5 fold increase in growth cone velocity (Kuhn et. al., 1995). This study, and our in vivo pathfinding results indicate that at decision points local environmental cues may induce growth cones to change migration rate and direction. Alternative models of growth cone guidance Our results indicate that differential filopodial adhesion to the extracellular environment is insufficient to direct T i l growth cone pathfinding events. Thus, what alternative cellular mechanisms could guidance molecules initiate to induce growth cone steering events in vivo? Evidence from in vitro studies suggests that the binding of filopodial receptors to extracellular matrix molecules may produce intracellular signalling cascades that modulate growth cone pathfinding. Many intracellular regulatory mechanisms, including kinases and phosphatases (Brambilla and Klein, 1995; Chang et. al., 1995; Desai et. a l , 1996; Krueger et. al., 1996; Gallo et. al., 1997; He et. al., 1997), calcium concentration (Williams et. a l , 1992; Gomez et. al., 1995; Davenport et. al., 1996, Kuhn et. al., 1998), PLCy (Saffell et. al., 1997), cAMP (Kim and Wu, 1996; Song et. al., 1997), and the small GTP-binding proteins (Kuhn et. al., 1997; Luo et. al., 1997; Hall, 1998) have been implicated in growth cone motility. In addition, it has been demonstrated that the concentration of substratum-bound ligand can post-translationally regulate the amount of receptor expressed on the surface of neurons (Condic and Letourneau, 1997). Recently, Renaudin et al. (1999) have used immunocytochemistry and confocal microscopy to demonstrate that p i integrins colocalize with vinculin at point contacts, and these adhesion sites are associated with various signalling proteins such as neuron specific FAK+, RhoA, RhoB and CDC42. Therefore, although much is yet to be learned about the relationships between local 57 second messenger cascades and subsequent changes in cytoskeletal organization and membrane adhesion, second messenger systems may regulate the assembly and interaction of force-generating machinery within pathfinding growth cones. 58 III. DISCRETE ROLES FOR SECRETED AND TRANSMEMBRANE SEMAPHORINS IN NEURONAL GROWTH CONE GUIDANCE IN VIVO 59 Introduction The establishment of accurate neuronal connectivity is a sequential process involving directed axon initiation, growth cone pathfinding, target selection, and finally synapse formation. During each of these stages neuronal growth cones are guided by many mechanisms, including both attractive and repulsive guidance molecules (reviewed by Goodman, 1996; Tessier-Lavigne and Goodman, 1996). Several gene families contain members that have been implicated in growth cone guidance, and it is becoming clear that guidance decisions are influenced by a balance of attractive and repulsive signals recognized by migrating growth cones (Stoeckli, 1997; Winberg et al., 1998). Proteins belonging to the semaphorin gene family have been demonstrated to function in both vertebrate and invertebrate nervous systems to mediate axon pathfinding and target selection (Culotti and Kolodkin, 1996; Mark et al., 1997). This large family of guidance molecules, comprised of both transmembrane and secreted glycoproteins, is characterized by a conserved -500 amino acid extracellular semaphorin (sema) domain. The first functionally characterized semaphorins were grasshopper semaphorin l a (g-Sema la, formerly Fasciclin IV), a transmembrane semaphorin shown to be essential for correct neuronal pathfinding in the developing grasshopper limb bud (Kolodkin et a l , 1992), and Collapsin-1 (Coll-1), a secreted semaphorin in chick shown to collapse dorsal root ganglion growth cones in vitro (Luo et al., 1993). Since then as many as 30 semaphorins, which can be subdivided into at least 7 structurally distinct classes, have been identified in many animal species from worms to mammals (Mark et al., 1997). Secreted semaphorins are characterized by the conserved sema domain, an immunoglobulin (Ig) domain C-terminal to the sema domain, and in vertebrates a carboxy terminal basic domain. Results from a variety of systems have demonstrated that these proteins 60 can function as inhibitory neuronal guidance cues (reviewed by Mark et al., 1997). Evidence implicating secreted semaphorins in chemorepulsion came initially from the finding that chick Coll-1 can cause the collapse of chick dorsal root ganglion growth cones in vitro (Luo et al., 1993). This collapse can direct growth cones to turn away from a source of Coll-1 by inducing a localized reorganization of the actin cytoarchitecture within the growth cone (Fan et al., 1993; Fan and Raper, 1995). Later studies showed that the mammalian orthologues of chick Coll-1, human Sema-III/mouse Sem D, could act as repellents to pattern sensory and motor axon projections in the spinal cord and brain (Messersmith et af, 1995; Behar et al., 1996; Puschel et al., 1996; Tanelian et al., 1997; Taniguchi et al., 1997; Varela-Echavarria et al., 1997). Further evidence for an inhibitory function for secreted semaphorins comes from invertebrate studies, where Drosophila semaphorin 2a (dSema 2a) has been shown to function as a selective target-derived cue capable of inhibiting synaptic arborization formation (Matthes et al., 1995; Winberg et al., 1998; the original D-Sema-II has been renamed dSema 2a, C. Goodman, i personal communication). Taken together, the results from invertebrate and vertebrate studies suggest that the secreted semaphorins are capable not only of inducing growth cone collapse but also of directing growth cone pathfinding, target selection and synaptic terminal arborization. The role of transmembrane semaphorins in neuronal development is less well characterized. Similar to secreted semaphorins, genetic analysis has shown the transmembrane Drosophila semaphorin dSema l a acts as a repellent and is important for the guidance of CNS and motor neurons (Yu et a l , 1998). In vivo antibody perturbation experiments revealed that g-Sema la, a transmembrane semaphorin expressed in the developing grasshopper limb bud, is important for establishing the peripheral T i l pioneer projection into the CNS; however, it remains unclear whether it performs this role as a repellent or an attractant (Kolodkin et al,. 1992). In addition, evidence suggests that Sema l a acts as an attractive guidance cue for later arising neurons in the limb bud (Wong et al., 1997). 61 Genetic analysis in Drosophila demonstrates that the invertebrate secreted semaphorin dSema 2a acts in combination with a variety of other molecules, including Netrin B and Fasciclin II, to inhibit promiscuous synaptogenesis and to provide a repulsive force for motor neuron growth cones at specific choice points. This clearly demonstrates that growth cones integrate multiple signals during pathfinding and target selection (Winberg et al., 1998). The presence of multiple signalling molecules and guidance cues in the developing grasshopper limb during the period of T i l pioneer axon outgrowth provides a powerful model system for the investigation of functional interactions among these cues (reviewed by Sanchez et al., 1995). Therefore, to elucidate the interaction between different members of the semaphorin family on the guidance of a single well-characterized neuron projection, we have identified and characterized a novel secreted semaphorin in grasshopper, gSema 2a. During the period of T i l pioneer axonogenesis and extension toward the CNS, gSema 2a is expressed in a striking gradient by the epithelium of the developing limb bud. Our in vivo antibody perturbation results demonstrate this is a chemorepulsive gradient, critical during several key stages of the T i l pioneer pathway. Initially, Sema 2a chemorepulsion is required for the reliable establishment of proximal T i l axonogenesis; thereafter, the gradient of Sema 2a directs growth cone pathfinding, maintains fasciculation and ensures continued proximal extension towards the CNS. Secreted semaphorins can, therefore, contribute to both the initial stages of directed axon outgrowth as well as subsequent growth cone pathfinding events. We further show that in vivo antibody perturbation of both secreted Sema 2a and transmembrane Sema l a results in novel T i l axon defects and an increased incidence of defects. This is the first study to demonstrate that different members of the semaphorin family can provide independent guidance information to the same neuronal growth cone in vivo. Therefore, at all stages of outgrowth and pathfinding, growth cone decisions are based on the integration of multiple guidance signals. 62 Materials and Methods PCR Methods, cDNA Isolation, and Sequence Analysis cDNA to poly(A)+ R N A from grasshopper embryos at -45% of development was prepared and used at lOng per 100 pi PCR reaction. PCR was performed using Taq polymerase (Saiki et al., 1988) and the partially degenerate oligonucleotides G C G A A T T C T T [CT] TT [CT] TT [CT] C G N G A [ AG] A C N G C (corresponding to an Eco R l site and amino acids (aa) FFFRKTA) and G C G A A T T C T C C C A N G C [ G A ] C A [ G A ] T A N G G [ G A ] T C (corresponding to aa DPYCAW[D/E] and an Eco R l site). PCR cycling conditions were: 40 cycles of 94°C for 1.5 min, 46°C for 1.5 min, 72°C for 2.5 min, followed by one cycle of 72°C for 10 min. A 700 bp product was obtained, subcloned into pBluescript (Stratagene), sequenced on both strands using a Perkin Elmer Applied Biosystems Division 373 a automated D N A sequencer and found to encode a portion of a novel semaphorin domain. This amplification product was used to screen 1.25 X 10^ clones from a Xgtl 1 grasshopper embryonic cDNA library (Snow et al., 1988). A single positive cDNA clone was isolated, sequenced in its entirety, and found to encode an incomplete open reading frame (ORF) for Sema 2a. Subsequent screening of a second embryonic grasshopper XZAP (Stratagene) cDNA (Seaver et al., 1996) library using this clone resulted in the isolation of ~20 positive clones. One clone was sequenced on both strands over the Sema 2a ORF and immediately flanking 5' and 3' untranslated regions. This clone was used for all subsequent analyses and cloning. In Situ Hybridization and Northern Blot Analysis Nonradioactive, digoxygenin (DIG-11-UTP)-labelled cRNA probes with either sense or antisense orientation were synthesized by run-off in vitro transcription using T3 and T7 R N A polymerases (Boehringer Mannheim). Probes were generated from a 700 base pair region of the 63 conserved sema domain of Sema 2a. In situ hybridization for Sema 2a was performed using the above described digoxygenin-labeled cRNA antisense and sense riboprobes on whole fixed embryos. Schistocerca gregaria embryos at developmental stages 30 - 45% were dissected in phosphate buffered saline (PBS), fixed for 50 minutes in P E M - F A (0.1 M PIPES [pH 6.95], 2.0 m M EGTA, 1.0 m M M g S 0 4 , 3.7% formaldehyde), washed for 1 hour with three changes in PBT (1XPBS, 0.5% Triton X-100, 0.2% bovine serum albumin), then incubated for 5 hrs at room temperature in prehybridization buffer (50% formamide, 5XSSC, 5X Denhardt's, 250 pg/ml Baker's Yeast RNA, 500 pg/ml sheared Herring Sperm DNA). Prehybridization buffer was replaced with hybridization buffer (fresh prehybridization buffer plus 500 ng/ml sodium carbonate hydrolyzed digoxygenin-labeled cRNA probe, which was previously heated to 80 degrees and placed on ice), and incubated overnight at 53 °C in a rotating incubator. Following overnight hybridization, non-hybridized probe was removed by first rinsing in 5XSSC at room temperature for 10 minutes, washed in 0.2XSSC for 60 minutes at 53°C, followed by a final wash in 0.2XSSC at room temperature for 10 minutes. At room temperature, embryos were then rinsed in maleic acid buffer (0.1 M Maleic acid, 0.15 M NaCl pH 7.5) for 5 minutes, blocked for 60 minutes (blocking buffer: 1% milk powder in 0.1 M Maleic acid, 0.15 M NaCl pH 7.5), and incubated with anti-digoxygenin antibody 1:10000 dilution in blocking buffer for 60 minutes. Embryos were rinsed twice for 30 minutes with maleic acid buffer, equilibrated in Tris saline (100 m M Tris-HCl pH 9.5, 100 m M NaCl, 5 m M MgCl 2 ) for 5 minutes, and incubated overnight in Tris saline plus 0.34 mg/ml N B T and 0.18 mg/ml BCIP. For overnight incubation eppendorf tubes were wrapped in aluminum foil and placed on a rocking platform. The reaction was stopped by rinsing 3X with PBS, and then incubated for 30 minutes in PBS. Embryos were cleared with 70% glycerol, and mounted in 90% glycerol. For Northern blot analysis, total R N A was extracted from stage 35% embryos using Trizol (Gibco BRL) , separated by electrophoresis through a 1.1% agarose-formaldehyde gel, 64 blotted onto Hybond-N+ (Amersham), and hybridized with either the digoxygenin-labeled cRNA probe used for in situ hybridization or a random primed P 3 2-dATP-labeled D N A probe corresponding to the same 700 base pair region. GSema 2a Antisera Production and Immunoblot Analysis Immunizing rabbits with 6-histidine-tagged Sema 2a fusion proteins that were expressed in E. coli produced anti-grasshopper Sema 2a polyclonal antibodies. The bacterial expression constructs were made by PCR amplification of each fragment of Sema 2a and inserted into the Hindlll and SphI sites of pQE30 (Qiagen). Expressed protein was purified by immobilized nickel-chelate affinity chromotography (Qiagen). The fusion proteins of Sema 2a corresponded to the following 5 different regions; amino acids 32-151 (SD1; 13kD N-terminal sema domain peptide), amino acids 152-393 (SD2; 26kD middle sema domain peptide), amino acids 394-506 (SD3; 13kD C-terminal of sema domain peptide), amino acids 516-697 (CT1; 20kD C-terminal and Ig domain peptide), and amino acids 625-697 (CT2; 7kD C-terminal peptide). In total, 9 rabbits were injected with Sema 2a fusion proteins. Two antibody stocks were generated against SD1, one against SD2, and two against SD3; within the C-terminus, two antibody stocks were generated against CT1, and two* were generated against the fusion protein CT2. IgG antibodies were purified by protein A-Sepharose chromatography (Pierce) and Fab fragments were generated using ImmunoPure Fab Preparation Kit (Pierce). A l l antibodies were stored at -20°C. For blocking experiments, antibodies were dialyzed against RPMI and stored at -20°C. For Western blot analysis, grasshopper embryonic stage 30-40% whole-cell lysate was probed with the various Sema 2a antibodies (1:1000) and reacted for E C L chemiluminescence as described by the manufacturer (Amersham). 65 Immunocytochemistry Schistocerca Gregaria embryos at developmental stages 30 - 40% were dissected, fixed and washed as above for in situ hybridization, and then incubated overnight at 4°C in primary antibody. For Sema 2a staining, whole sera and purified IgGs were diluted 1:500 in PBT; for neuronal staining, rabbit and goat anti-HRP (Jackson Immunoresearch Lab) were diluted 1:1000 in PBT. The embryos were washed for 1 hr in PBT with three changes, and incubated in the appropriate secondary antibody (1:250 dilution in PBT) for 2 hours at room temperature. Following secondary incubation, embryos were washed for 1 hour with 6 changes of PBT. Fluorescently labelled embryos were mounted in Slowfade antifade reagent (Molecular Probes) and viewed under fluorescence microscopy. .For Sema 2a/Sema l a double-labelling, embryos were incubated simultaneously in the two primary antibodies overnight (1:500 Sema 2a, 1:1 Sema l a Mab 6F8), the embryos were washed, and the Sema l a antibody 6F8 visualized by incubating in secondary antibody Cy3 conjugated goat anti-mouse (1:250) for 2 hours at room temperature, followed by washing and then visualization of Sema 2a antibody by incubating in secondary antibody FITC conjugated goat anti-rabbit (1:250) for 2 hours at room temperature. The embryos were washed and mounted as above. For Sema 2a and neuronal double labelling, embryos were labeled sequentially for anti-HRP followed by Sema 2a. Antibody blocking experiments For all blocking experiments, dialyzed IgGs and Fab fragments were diluted into freshly made supplemented RPMI culture media plus 10% FBS (Wong et. al., 1997). Prior to culturing, embryos from a single clutch were sterilized, dissected and the entire amnion and dorsal membrane removed from the embryo to ensure access of the antibodies during culturing. As embryos within a single clutch typically differ by less than 1% of embryonic development 66 (Bentley et al., 1979), each clutch was randomly divided into experimental and control groups, with several embryos fixed immediately to determine developmental stage at the start of culturing. Embryo culturing began at 30% of development, just prior to T i l pioneer neurons axonogenesis. Following approximately 30 hours in culture embryos were fixed and immunostained with antibodies to HRP (Jan and Jan, 1982) for visualization of T i l axons and other neurons. For each culture experiment, limbs from antibody cultured embryos were compared with limbs from non-antibody cultured and preimmune-antibody cultured embryos. For embryos incubated in Sema 2a alone, the T i l pathway was scored as abnormal for one or more of the following observed characteristics: defasciculation for a minimum distance of 50pm anywhere along the pathway; dorsal projection of one or both axons within the femur for a minimum distance of 25pm; distal projection of one or both axons; failure of one or both axons to turn ventrally at the trochanter-coxa segment boundary, typically characterized by continued proximal extension towards the CNS; or dorsal extension toward the Cx2 cells. For embryos incubated in Sema l a alone, the T i l pathway was scored as abnormal for one or more of the following observed characteristics (Kolodkin et al., 1992): defasciculation for a minimum distance of 25pm anywhere along the pathway; multiple axon branches that extended ventrally within the trochanter; axon branches crossing the trochanter-coxa boundary dorsal to the C x i cells; and axon branches that crossed the trochanter-coxa segment boundary, did not turn ventrally, but continued proximally toward the CNS. For embryos incubated in both Sema 2a and Sema la, the T i l pathway was scored as abnormal i f it met any one of the criteria described above for either Sema 2a or Sema la. The novel hybrid phenotype defects observed were characterized by defasciculation anywhere along the pathway accompanied by both extensive axonal branching as well as distal or dorsal projection abnormalities. In the hybrid phenotypes, a variety of dorsal projection defects were observed including continued proximal extension in the 67 dorsal compartment; extensive fasciculation with the Cx2 cells; or a ventral turn within the coxa to contact the C x i cells. For each antibody tested, the data are presented as a percentage of the abnormal T i l pathways observed. Error bars = S E M , calculated using the number of experiments and their average observed aberrance; n = number of limbs scored for each treatment. 68 Results Identification of a Novel Secreted Grasshopper Semaphorin Gene The conservation of the 500 amino acid sema domain among members of the semaphorin gene family enabled us to use a degenerate PCR-based strategy to isolate a new secreted grasshopper semaphorin, called gSema 2a (see Experimental Procedures). Sema 2a is a secreted protein that is most similar to Drosophila semaphorin 2a (dSema 2a), with which it shares 67% amino acid identity within the sema domain and 63% within the C-terminal (Fig. 10). Grasshopper Sema 2a contains a semaphorin domain and an Ig domain; however, similar to dSema 2a, gSema 2a does not have the basic C-terminal domain observed in the vertebrate homologues. Grasshopper Sema 2a shares approximately 34% and 23% amino acid identity with human Sema-III/mouse Sem D/chick Coll-1 within the sema and C-terminal domain, respectively. A comparison of the secreted semaphorin gSema 2a and the transmembrane grasshopper semaphorin Sema l a reveals the two grasshopper semaphorins share 37% amino acid identity within the sema domain. Initial characterization of Sema 2a by Western blot analysis, using antibodies generated against three regions of the sema domain (SD1-3) and two regions of the C-terminal domain (CT1 and CT2) (Fig. 11 A), detected a protein at approximately 80kD in grasshopper lysate (Fig. 1 \B). Duplicate antibodies from independent rabbits were generated against all regions except SD2, and all antibodies detected the same 80kD band. Pre-immune sera revealed no bands at 80kD (Fig. 115) and none of the Sema 2a antibodies cross-react with Sema l a (Fig. 1 I Q . Northern blot hybridization using either a Dig-labelled or a P3 2-labelled probe revealed a single transcript of approximately 5 kb (Fig. 1 ID). 69 Figure 10. Amino acid comparison of gSema 2a and dSema 2a. Amino acid sequence alignment between gSema 2a and dSema 2a. Identical amino acids are shown in bold, signal sequences in italics, and the Ig domains are underlined. The beginning and end of the semaphorin domains are indicated by a # symbol. Database accession no.: AF134904. 70 g S e m a 2 a MAAKLWNLLLVAASVHLVGSVEQhUQDhIHE 31 d S e m a 2 a MSLLQLSPLLALLLLLCSSVSETAADYENTWNFYYERPCCTGNDQGNNNYGKHGADHVRE 6 0 g S e m a 2 a FSCGHKYYRTFHLDEKRESLYVGALDKVYKLNLTNISLSDCERDSLTLEPTNIAN— -CVSK 90 d S e m a 2 a FNCGKLYYRTFHMNEDRDTLYVGAMDRVFRVNLQNISSSNCNRDAINLEPTRDDWSCVSK 121 g S e m a 2 a GKSADFDCKNHIRVIQPMGDGSRLYICGTNAHSPKDWVVYSNLTHLQRHEYVPGIGVGIAK 151 d S e m a 2 a GKSQIFDCKNHVRVIQSMDQGDRLYVCGTNAHNPKDYVIYANLTHLPRSEYVIGVGLGIAK 182 g S e m a 2 a CPFDPEDSSTAVWVENGNPGDLPGLYSGTNAEFTKADTVIFRTDLYNLTTGRREYSFKRTL 212 d S e m a 2 a CPYDPLDNSTAIYVENGNPGGLPGLYSGTNAEFTKADTVIFRTDLYNTSAKRLEYKFKRTL 243 g S e m a 2 a KYDSKWLDNPNFVGSFDVGEYVLFFFRETAVEYINCGKSVYSRVARVCKKDVGGKNILSQN 273 d S e m a 2 a KYDSKWLDKPNFVGSFDIGEYVYFFFRETAVEYINCGKAVYSRIARVCKKDVGGKNLLAHN 3 04 g S e m a 2 a WATFLKARLNCSIPGEFPFYFNEIQGVYK-MPNTDKFFGVFSTSVTGLTGSAICSFTLKDI 333 d S e m a 2 a WATYLKARLNCSISGEFPFYFNEIQSVYQLPSDKSRFFATFTTSTNGLIGSAVCSFHINEI 365 g S e m a 2 a QEVFSGKFKEQATSSSAWLPVLPREVPDPRPGECVNDTELLPDTVLNFIRSHPLMDGAVSH 3 94 d S e m a 2 a QAAFNGKFKEQSSSNSAWLPVLNSRVPEPRPGTCVNDTSNLPDTVLNFIRSHPLMDKAVNH 426 g S e m a 2 a EGGKPVFYKRDVLFTQLWDKLKVNLVGKNMEYIVYYAGTSTGQVYKWQWYDSGGLPQSL 4 55 d S e m a 2 a EHNNPVYYKRDLVFTKLVVDKIRID-IL-NQEYIVYYVGTNLGRIYKIVQYYRNGESLSKL 485 g S e m a 2 a LVDIFDVTPPEPVQALHLSKEYKSLYAASDNIVRQIELVMCHHRYSNCLQCARDPYCGWDR 516 d S e m a 2 a _ L-DIFEVAPNEAIQVMEISQTRKSLYIGTDHRIKQIDLAMCNRRYDNCFRCVRDPYCGWDK 545 # g S e m a 2 a DSNSCKSYNPGLLQDVTN-TSANLCEHSVMKKKLIVTWGQSIHLGCFLKVPEVLSSQTISW 576 d S e m a 2 a EANTCRPYELDLLQDVANETS-DICDSSVLKKKIWTYGQSVHLGCFVKIPEVLKNEQVTW 605 g S e m a 2 a VHYTKDKGRYPIVYRPDKYIETSEHGLVLISVTDSDSGRYDCWLGGSLLCSYNITVDAHRC 637 d S e m a 2 a YHHSKDKGRYEIRYSPTKYIETTERGLVWSVNEADGGRYDCHLGGSLLCSYNITVDAHRC 666 g S e m a 2 a SAPGGSNDYQKIYSDWCHEFERSKIAMKTWERKQAQCSTKQNNS-NQKTHPNDIFHSNPVA 697 d S e m a 2 a TPPNKSNDYQKIYSDWCHEFEKYKTAMKSWEKKQGQCSTRQNFSCNQ- -HPNEIFRKPNV 724 71 Figure 11. Recombinant peptides generated for antibody production, Western and Northern blot analysis. A, Schematic representation of the regions of gSema 2a used to generate antigenic fusion proteins; Sema Domain 1 (SD1), Sema Domain 2 (SD2), Sema Domain 3 (SD3), C-Terminus 1 (CT1) and C-Terminus 2 (CT2). See Experimental Procedures for amino acid number. B, Western blot analysis of whole-cell lysate from grasshopper embryos at 31% of development using the antibodies generated against the various regions of Sema 2a. A molecular weight of 80 kDa was consistently observed. PI, pre-immune. C, Western blot detection of a myc-tagged Sema l a fusion protein with Sema l a monoclonal antibody 6F8 (a-gSema la), monoclonal c-myc antibody and polyclonal antibodies to Sema 2a. None of the Sema 2a antibodies recognized Sema la. D, Northern blot revealed a single transcript of approximately 5kb. 72 73 Grasshopper Semaphorin Expression is Developmentally Regulated Sema 2a is expressed in the embryo prior to axonogenesis (<29%), and persists through 40% embryonic development, the latest stage we examined. At approximately 30% of development, a gradient of Sema 2a is evident in the limb bud epithelium with the highest protein distribution occurring in the distal and dorsal-most aspect of the limb (Fig. 12^4). This gradient of Sema 2a expression coincides with the differentiation and subsequent axonal projection of the well-characterized T i l pioneer neurons (which occurs between 30 - 35% of development), which establishes the first peripheral projection into the CNS from the limb (Fig. 13A, 5, F). Sema 2a does not appear to be expressed by the T i l neurons or other limb pre-axonogenesis neurons during this stage of development. By approximately 32% of development, as the T i l growth cones approach the trochanter segment epithelium, transmembrane Sema l a expression is also evident within the developing limb bud and is localized to a circumferential band in the trochanter (Fig. 135, Q . While Sema 2a and Sema l a are both expressed in the dorsal trochanter epithelium, little or no coexpression is observed elsewhere in the limb at this stage (Fig. 13Q. At approximately 33% of development, Sema 2a expression remains high in the distal and dorsal limb bud; however, increasing expression is also observed in the ventral epithelium distal to the trochanter Sema l a band (Fig. 125). By this stage (33%), the T i l growth cones have migrated proximally out of the Sema 2a gradient and have contacted the Sema l a expressing trochanter epithelium, where they make a stereotyped ventral turn (Fig. 13F). Sema 2a expression at developmental stages after the completion of the T i l projection (>35%) is localized to epithelial bands in the distal femur, mid to distal half of the tibia and the distal tip of the tarsus (Fig. 12C, D, E; Fig. 13D, E, F). Interestingly, the circumferential epithelial expression pattern of Sema 2a is, for the most part, complementary to the circumferential epithelial expression of 74 Figure 12. Sema 2a is expressed in the developing limb bud during Til pioneer axon outgrowth. A , Immiinocytochemistry at 30% of embryonic development reveals that Sema 2a is expressed throughout much of the limb bud epithelium. A gradient is evident, with the highest concentration of Sema 2a localized to the distal and dorsal aspect of the limb bud. B, At approximately 33% of development, Sema 2a expression is still high in the dorsal and distal regions of the limb. A n increase in expression is also apparent in the ventral region of the limb. C, By 34% of development, Sema 2a becomes restricted to circumferential bands of epithelial staining. The most proximal edge of Sema 2a expression ends in the proximal femur, near the femur-trochanter segment boundary. D, By 35% development, epithelial bands are evident in the mid to distal femur, distal half of the tibia and the distal tip of the tarsus. At this stage presumptive muscle staining is apparent in the developing limb (arrow demarcates extensor tibiae in dorsal femur). Sema 2a is heavily expressed in pluripodia (asterisk). E, Circumferential epithelial band expression pattern is still apparent at 38% of development. F, Dig-RNA antisense labeling of a limb at 38% of development revealed Sema 2a mRNA localization is identical to protein expression pattern in the developing limb bud. Circumferential bands are apparent in the mid to distal femur, tibia and tarsus, similar to the antibody labeling (E). G, Dig-RNA sense strand in situ hybridization of a limb at approximately 35% of development. H and /, In situ hybridization with Dig-RNA antisense shows Sema 2a expression in laminar cells of the eye (arrows) and circumferential epithelial bands in the antennae. For all limb figures, distal is to the left and dorsal is up. Immuncytochemistry using antibodies generated against SD1 is shown (A - E); however, the same staining pattern was seen with all antibodies. Arrow heads demarcate segment boundaries. Ta: Tarsus; Ti : Tibia; Fe: Femur; Tr: Trochanter. Scale bar is 50pm in A , 60pm in B and C, 65pm in D and G, 70pm in E, F , H and I. 75 Figure 13. Sema 2a and Sema l a expression during Til pioneer axon outgrowth. A and B, Sema 2a and anti-HRP double staining at 30% and 32% development, respectively (green: Sema 2a protein; red: HRP-stained neurons; arrow heads demarcate the region of the trochanter limb segment; arrows indicate the Cxi cells located proximal to the Tr limb segment). C, Sema 2a and Sema la double staining at 32% embryonic development. Sema 2a and la are both expressed in the dorsal trochanter (arrows), (green: Sema 2a; red: Sema la; arrow heads demarcate the region of the trochanter limb segment). D and E, Sema 2a and Sema la double staining at 35% and 38% embryonic development, respectively, (green: Sema 2a; red: Sema la; arrowheads demarcate limb segment boundaries; Ta: tarsus; Ti: tibia; Fe: femur; Tr: trochanter). F, Schematic of the Sema 2a and Sema la expression pattern during Til outgrowth (31%) panel: Til: Til pioneer neurons; Tr: trochanter guidepost neuron; Cxi: coxa 1 guidepost neurons; 35% panel: arrow heads demarcate segment boundaries; Ta: tarsus; Ti: tibia; Fe: femur; Tr: trochanter; Cx: coxa). Scale bar is 40pm in A, 50pm in B, C, 55pm in D, and 100pm in E. 77 78 transmembrane Sema l a (Fig. 13D, E, F). In general, within each limb segment Sema 2a is expressed in circumferential bands in the mid to distal region of each segment, while Sema l a is expressed in the proximal region (Fig. 13D, E). The spatial and temporal distribution of Sema l a and 2a suggests that these molecules may be involved in the guidance of a variety of neurons that arise during limb bud development. In addition to the epithelial expression, Sema 2a is expressed by presumptive muscle precursor cells within the developing limb bud and throughout the entire embryo (Fig. \2D), laminar cells within the developing eye (Fig. \2H) and in circumferential epithelial bands of the antennae (Fig. 127). In particular, high levels of Sema 2a expression are evident in the precursor muscle cells of the extensor tibiae and the levator and depressor tarsus, two large muscle groups in the developing limb bud. Interestingly, pan muscle expression of secreted dSema 2a was observed in Drosophila embryos, and analysis of well-understood muscle populations revealed dSema 2a can act as an inhibitory guidance cue necessary for accurate motor neuron target selection and synapse formation (Winberg et al., 1998). At all stages examined, the distribution of Sema 2a protein detected with the different antisera corresponds identically to the sites of Sema 2a gene expression as detected by in situ hybridization (Fig. 12F, G). Furthermore, all antibodies generated against the different regions of Sema 2a produced the same immunocytochemistry results, including experiments using duplicate sera generated from independent animals and C-terminal antibodies. 79 Antibodies Directed Against the Conserved Sema Domain of gSema 2a Disrupt Til Pioneer Axon Pathfinding The striking gradient of Sema 2a protein in the developing limb bud during the period of T i l pioneer neuron axonogenesis and outgrowth suggests that Sema 2a directs growth cone guidance at this location. To investigate the function of Sema 2a, we cultured grasshopper embryos in the presence of Sema 2a antibodies during the 5% of development necessary for the establishment of T i l pioneer projection into the CNS. For each culture experiment, we compared the percentage of aberrant pathways observed following incubation in RPMI media alone with that observed following the addition of Sema 2a purified IgG, Fab fragments or purified IgG isolated from preimmune sera. Given that all the Sema 2a antibodies generated (including duplicate sera from independent animals) demonstrated the same high degree of specificity for Sema 2a protein on Western blots and the same immunocytochemistry results, all antisera were tested for function blocking activity. Under normal conditions the T i l neurons establish a stereotyped projection through the limb bud into the CNS. Typically the growth cones of the T i l neurons extend proximally along the femur epithelium until they contact and reorient toward a pre-axonogenesis neuron (Tr cell) located in a circumferential band of Sema l a expressing epithelium (Fig. 13^ 4, B, C, F). The growth cones then extend ventrally along the Sema l a expressing epithelium before contacting and reorienting toward another pre-axonogenesis neuron (Cxi) located in the coxa. After contact with the C x i cells, the growth cones extend proximally into the CNS. The two T i l axons typically remain fasciculated along the entire length of the projection. For the majority of antibody perturbation experiments we dissected and cultured embryos at approximately 30% of development, the stage just prior to T i l pioneer neuron axonogenesis (Fig. 13^ 4 and Fig. 144). Embryos cultured in the presence of culture media alone, preimmune antiserum IgG, or l p M CT2 antiserum IgG or Fab fragments, had no observable effect on T i l axon pathfinding 80 Figure 14. Antibodies directed against the semaphorin domain of Sema 2a disrupt Til pioneer pathfinding. A, Embryos fixed at the start of the experiment, (Time 0: approximately 30% of development), reveal T i l pioneer neurons have just differentiated from the underlying epithelium. B, The T i l pioneer projection is normal following 30 hours incubation in RPMI culture media. C - F, Four representative examples of limb buds following 30 hours of culturing in RPMI media in the presence of antibodies directed against the semaphorin domain of Sema 2a (1 uM). C, This limb shows a distal projecting phenotype following antibody perturbation of the SD2 region. Asterisk indicates the T i l cell bodies, arrows demarcate distal and proximal projecting growth cones. D, Early defasciculation and dorsal projection seen in a limb following incubation with SD1 antibodies. One growth cone has crossed the trochanter into the dorsal coxa and the second growth cone has projected correctly to the C x i cells in the ventral coxa. E and F, Two examples of the dorsal looping axon phenotype. Arrows demarcate growth cones that have wrapped around dorsally and are in contact with (E), or near (F), the T i l cell bodies. Both limbs are from SD2 antibody blocking experiments. G, Antibodies generated against the last 7kD of Sema 2a, the region C-terminal to the Ig domain, do not disrupt T i l axon pathfinding (1 uM). H, A typical limb following incubation in the presence of 1 u M SD2 pre-immune, demonstrating pre-immune antibodies do not disrupt T i l axon pathfinding. In all panels, dorsal is up and distal is to the left; arrow heads indicate the region of the trochanter segment. Scale bar is 50pm in A , C, D, E, and F, is 60pm in H, and is 65pm in G and B. 81 (Fig. 145, G, H, Fig. 15). In contrast, embryos cultured in the presence of antibodies directed against the semaphorin domain (SD1-3) exhibited a number of aberrant projections that typically fell into three classes. These include: 1) the direct distal growth of one of the T i l axons (Fig. 14C); 2) defasciculation of the two pioneer neuron axons along the entire length of the femur, followed by the dorsal projection of at least one of the axons (Fig. 14D); and 3) dorsal and distal growth of at least one of the axons (Fig. 14E, F). Often the dorsal projecting axons looped completely around and the growth cones would be in close proximity to their cell bodies (Fig. \AE, F). Considering the high expression of Sema 2a in the dorsal and distal tip of the limb bud during the period of T i l pioneer neuron axonogenesis, the nature of the guidance errors are consistent with a repulsive role for the gradient of Sema 2a. Interestingly, although dorsal projection abnormalities were common, we rarely observed axons aberrantly projecting into the ventral limb compartment. This suggests that, normally, the chemorepulsive gradient of Sema 2a is critical for counterbalancing an attractive distal/dorsal cue or possibly a repulsive ventral cue. In control cultures, aberrant projections were observed in approximately 10% of limbs, establishing the baseline level of T i l projection errors for these cultures (Fig. 15). These control projection abnormalities were subtle and typically included defasciculation and aberrant dorsal projections within the trochanter limb segment. Furthermore, increasing the concentration of the CT2 antibodies up to 2 p M (data not shown), or the preimmune antiserum to 2 u M (Fig. 17; page 91), had no effect on the number or type of guidance errors. Embryos cultured in the presence of antisera generated against the entire C-terminus including the Ig domain (CT1) did, however, lead to increased frequency of T i l axon projection abnormalities (Fig. 15). Yet, even using the highest concentration of CT1 antibody (l.OpM), the frequency of errors was 50% lower than observed in the semaphorin domain perturbation experiments. Moreover, the type of abnormalities observed following the l.OuM CT1 blocking experiments were similar to control 83 Figure 15. Summary of Sema 2 a antibody perturbation experiments. Embryos cultured in the presence of antibodies generated against the sema domain of Sema 2a exhibit a dose-dependent increase in the frequency of aberrant T i l projections over both control and C-terminal perturbation experiments. Furthermore, perturbation of the SD2 region of the sema domain, at both l p M and 0.5pM, led to a significantly higher incidence of defects compared to either SD1 or SD3 antibody perturbation experiments. Data are pooled from 18, 20, 16, 10 and 11 experiments using antibodies directed against SD1, SD2, SD3, CT1 and CT2, respectively, n = number of limbs scored for each treatment. 84 c/) CO | -i—» 03 6 0 4 0 2 0 0 6 0 4 0 c 2 o 6 0 4 0 2 0 0 CD _ Q CO CM in _3. CO CO SD1 _I_ T o *" SD2 SD3 ^ ^ ^ 6 0 4 0 2 0 0 6 0 4 0 2 0 0 CT1 T CN CM CO T — T — , It] CT2 i CO J L o CM CM —UsJ— r--* # <?\<5> s Fab IgG Controls 85 abnormalities, typified by defasciculation and dorsal projection within or near the trochanter limb segment. In contrast, addition of sema domain antiserum IgG or Fab fragments to the culture medium at concentrations between 0.1 - l .OpM resulted in a dose response disruption of the normal T i l axonal projection (Fig. 15). Of the three general classes of defects, defasciculation and dorsal projection abnormalities were most commonly observed; however, all three classes of malformations occurred more frequently than in the control experiments. For example, we rarely observed the distal or dorsal looping projections in the control experiments, or in the experiments employing antibodies that recognize the C-terminus (CT1 and CT2). In addition, following sema domain perturbation, the defasciculation and aberrant dorsal projection phenotypes often occurred distal to the trochanter limb segment; whereas, following blocking of the C-terminus or in the control experiments, these phenotypes were observed most often near or within the trochanter segment. Although the frequency of pathfinding errors varies with concentration, it remains undetermined whether the distribution of abnormal phenotypes is also dose-dependant. Culturing in the presence of Fab or IgG antibodies showed similar results with no significant variation in frequency of malformed T i l pathways or the type of defects observed (Fig. 15). Furthermore, perturbation studies using the duplicate sera showed statistically similar results, reflected in the calculated frequency of pathfinding errors illustrated in Fig. 15. Interestingly, following perturbation of the SD2 domain the distal and dorsal looping projection phenotypes were observed more often, and the frequency of aberrant projections was significantly greater for both the IgG and Fabs, than was observed following perturbation using antibodies directed against other regions of Sema 2a (p<0.05). Western analysis and immunocytochemistry results are identical for all the antibodies generated against Sema 2a. However, the CT1 and CT2 antibodies, including duplicate sera generated in independent animals, display little or no axon guidance blocking activity. These 86 results suggest that the C-terminus of Sema 2a is less critical for mediating the guidance function of the molecule, or alternatively, that none of the polyclonal antisera we have generated against the C-terminus recognizes a functionally important region. Our results demonstrate that the semaphorin domain, in particular the central region, plays an important role in mediating the axonal guidance activity of Sema 2a. gSema 2a and gSema l a Guide Discrete Regions of the Til Pioneer Projection At approximately 33% of development the T i l growth cones have migrated out of the gradient of Sema 2a in the proximal femur and have contacted the Sema l a expressing trochanter epithelial band, where they make a stereotyped ventral turn (Fig. 13F). By this stage of development, Sema 2a and Sema l a are both expressed by the dorsal trochanter epithelium (Fig. 13C), and although T i l growth cones frequently extend branches into this region, they normally retract, reorient and grow ventrally along the Sema l a expressing epithelium (O'Connor et al., 1990). Although perturbation of Sema 2a function after the T i l growth cones have reached the Sema 1 a expressing trochanter epithelium has no apparent effect on pathfinding, antibody perturbation of transmembrane Sema l a function at this later developmental stage does lead to T i l projection abnormalities (Fig. 165; Kolodkin et al., 1992). Typical Sema l a perturbation abnormalities include defasciculation and multiple branching of the two sibling pioneer axons within or near the Sema l a expressing trochanter epithelia, and also results in the formation of axon branches that extend proximally across that trochanter-coxa boundary. Therefore, it is likely that the chemorepulsive gradient of Sema 2a determines the initial direction of T i l axon outgrowth, maintains fasciculation, and ensures accurate projection to the trochanter; however, once the T i l growth cone contacts the trochanter epithelium, Sema la is responsible for maintaining axon fasciculation and preventing branching and extension into the coxa limb Figure 16. Sema 2a and Sema la act in combination to guide Til axon pathfinding. A , A typical Sema 2a antibody blocking phenotype, illustrating early axon defasciculation and dorsal projections. The growth cone of one of the T i l neurons has contacted the heavily labeled femoral chordontal organ (upper arrow). B, Typical Sema l a antibody blocking phenotype, illustrating axon defasciculation, branching within and near the Sema l a trochanter epithelium, and proximal extension into the coxa. C and D, Representative aberrant projections observed in combined Sema 2a and Sema l a antibody blocking experiments. Note both early defasciculation and dorsal projections, extensive axon branching along the entire projection, and proximal extension into the coxa. Phenotypes characteristic of the independent blocking of Sema 2a or Sema l a were also observed in the combination blocking experiments, see inset in Fig. 17. In all panels, arrow heads indicate region of trochanter segment. The scale bar is 50pm. 88 segment. It remains unclear whether Sema l a is acting as a repulsive guidance cue, restricting branching and defasciculation, or a permissive/attractive cue, preventing proximal extension past the trochanter. Regardless of the nature of Sema l a signalling in the trochanter, however, the results from blocking each semaphorin independently demonstrate that the two semaphorins guide discrete regions of the same axon pathway. Although the two semaphorins appear to have temporally, spatially and functionally distinct roles in T i l axon guidance, at approximately 32-33% of development the T i l growth cones interact with both the Sema 2a chemorepulsive gradient and the Sema 1 a expressing trochanter epithelium. To investigate the role of different semaphorin family members acting simultaneously on a single well-characterized axon projection, we cultured embryos in the presence of both Sema 2a and Sema l a antibodies: thus blocking both semaphorins during the period of T i l axon pathfinding. Similar to the Sema 2a perturbation experiments, stage 30-31% embryos were cultured for the duration of T i l growth cone pathfinding into the CNS. Consistent with our previous experiments we observed the typical early defasciculation and distal or dorsal axon projection in 56%) of limbs from embryos cultured in the presence of l u M Sema 2a SD2 IgG (Figs 16,4 and 17). Incubation with l p M Sema la monoclonal antibodies led to defects in 52% of limbs, again consistent with earlier experiments, these defect were typified by defasciculation and multiple branching of the two sibling pioneer axons within or near the Sema 1 a expressing trochanter epithelia, and axon branches that extend proximally across that trochanter-coxa boundary (Fig. 165 and Fig. 17) Unlike the Sema 2a perturbation phenotype, distal or dorsal looping abnormalities were not observed following Sema l a perturbation. Also, unlike the typical Sema l a phenotype, multiple branch formation was not observed following Sema 2a perturbation experiments. Control cultures (RPMI alone or 2 u M pre-immune) showed few aberrant projections, and the phenotypes were characteristic of baseline defects (Fig. 17). 90 Figure 17. Summary of combined Sema 2a and Sema l a antibody perturbation experiments. Graph illustrates percentage of aberrant T i l pathways following culture either in the presence of antibodies directed against the SD2 region of Sema 2a (56%), gSema l a (52%), both Sema l a and the SD2 region of Sema 2a (80%), SD2 pre-immune (13%) or RPMI culture media alone (14%). Inset shows the breakdown of phenotypes observed in combined Sema 2a / Sema l a blocking experiments. Of the 80 % aberrant T i l pathways, 30 % exhibited abnormalities characteristic of the independent perturbation experiments of Sema 2a, 23 % exhibited the independent Sema l a antibody perturbation phenotypes, and 27 % exhibited hybrid phenotypes. Data are pooled from 3 experiments in which 1 u.M SD2 Sema 2a IgG, 2pM SD2 pre-immune IgG and l p M monoclonal antibody 6F8 were used as blocking reagents, n = number of limbs scored for each treatment. 91 92 Following overnight culture in the presence of both 1 p M Sema 2a SD2 antibodies and 1 u M Sema l a antibody, embryos exhibited malformed T i l pioneer pathways in 80% of the limbs, a frequency substantially greater than observed following perturbation of either semaphorin alone (Fig. 17; p<0.05). The phenotypes included axon abnormalities characteristic of the independent Sema 2a and Sema l a perturbations experiments, such as embryos displaying either early defasciculation, distal and dorsal projection (30% of limbs; Fig. 17), or defasciculation within the trochanter and extensive axon branching (23% of limbs; Fig. 17). However, in addition to these previously characterized malformations, we observed new aberrant phenotypes that resembled hybrid forms of Sema 2a and Sema l a defects (Fig. 16C, D). Such T i l pathways exhibited early axon defasciculation, dorsal or distal projections, and extensive branching (27% of limbs; Fig. 17). Although we tested only one concentration of antibody (1 pM), it is noteworthy that the errors in the double-block experiment appear nearly additive. For example, following perturbation of both semaphorins 30% of limbs displayed Sema 2a phenotypes and 27% displayed a hybrid, this combined 57% is close to the 56% error observed following blocking Sema 2a alone. Similarly, 23% of limbs displayed Sema l a phenotypes and 27% displayed a hybrid, this combined 50% is close to the 52% error observed following blocking Sema la alone. The typical phenotypes observed in the independent and combined antibody perturbation experiments are summarized in Fig. 18. These results demonstrate that the perturbation of both Sema 2a and Sema l a within the developing limb bud leads to an increased frequency and range of abnormal T i l pioneer axon projections, illustrating that the combined functions of a secreted and transmembrane semaphorin can guide individual growth cones in vivo. Figure 18. Relationship of the Til projection in control and semaphorin antibody blocking experiments. A, Control (RPMI media, pre-immune or CT2 antibodies): Shading represents the graded distribution of Sema 2a. Bars represent Sema la distribution. The T i l axons extend proximally from the T i l cell bodies, turn ventrally in the Sema l a expressing trochanter epithelium, and subsequently contact the C x i cells and turn proximally toward the CNS. B-D, Schematics illustrating the range of observed phenotypes in the presence of Sema 2a function blocking antibodies. Axons defasciculate and migrate toward areas that exhibit high Sema 2a levels. Lighter shading represents blocking of Sema 2a function. E, Schematic illustration of typical phenotype observed in the presence of Sema 1 a function blocking antibody. Axons migrate normally to the trochanter, but branch and defasciculate in and near the region expressing Sema la, as well axon branches often project aberrantly into the coxa. However, aberrant T i l growth cones often eventually contact C x i guidepost cells and project successfully to the CNS. Lighter bars represent antibody blocking of Sema la. F, Schematic representation of a typical phenotype observed in the presence of both Sema 2a sema domain and Sema l a function blocking antibodies. T i l axons exhibit a range of aberrant phenotypes including early defasciculation with distal and dorsal projections (characteristic of Sema 2a blocking), defasciculation and branching within the trochanter and proximal extensions into the coxa (characteristic of Sema l a blocking) and combinations of these phenotypes. Lighter shading of Sema 2a and Sema la represents the blocking of function. 94 A Control B Sema 2a BLOCK C Sema 2a BLOCK D Sema 2a BLOCK E Sema la BLOCK Sema 2a & la BLOCK 9 5 Discussion Growth cone steering is a complex, highly integrated process involving the reception and transduction of multiple guidance cues into motile forces. The semaphorin gene family has been implicated in neuronal development in organisms ranging from Drosophila to human, with members displaying remarkable conservation phylogenetically both in sequence and function (Mark et al., 1997). In this study, we have characterized Sema 2a, a novel secreted semaphorin in grasshopper. We have used the embryonic grasshopper T i l pioneer pathway to demonstrate that this secreted semaphorin acts both independently and in combination with the transmembrane semaphorin Sema l a to ensure accurate axonal outgrowth and pathfinding in vivo. Our results illustrate that growth cones in vivo are influenced by the input from multiple guidance cues and steering decisions are based on the relative balance of these cues. A chemorepulsive role for gSema 2a in establishing the Til pioneer projection The establishment of the stereotypic T i l pioneer projection requires a series of growth cone pathfinding events beginning with the decision to extend proximally along the limb axis. This may be mediated, in part, by the plane of division of the epithelial cell that gives rise to the T i l neurons and to the expression of Fasciclin II in the surrounding epithelium (Lefcort and Bentley, 1989; Diamond et al, 1993). During T i l pioneer neurons axonogenesis there is a graded distribution of Sema 2a, with the highest expression in the distal and dorsal compartments of the limb. Antibody blocking of Sema 2a at this stage results in defects consistent with the T i l pioneer growth cones requiring Sema 2a to direct and maintain their proximal extension toward the CNS. For example, Sema 2a antibody perturbation during the early stages of T i l axonogenesis and outgrowth results in a significant number of axons aberrantly projecting into the distal tip and dorsal regions of the limb, areas expressing high levels of Sema 2a. Therefore, 96 T i l growth cones prefer to migrate down this gradient of Sema 2a protein; we believe this to be the first demonstration of an observable in vivo chemorepulsive gradient. gSema 2a similarity with other secreted semaphorins Sequence analysis reveals Sema 2a is most closely related to the Drosophila secreted semaphorin, dSema 2a and, similar to gSema 2a, during embryogenesis dSema 2a is expressed by muscles (Kolodkin et al., 1993; Winberg et al., 1998). Loss-of-function dSema 2a mutants have revealed specific targeting errors in which neurons make ectopic contacts with inappropriate muscles (Winberg et al., 1998). Correspondingly, gain of function dSema 2a mutants exhibit stalling of specific growth cones and failure to innervate target muscle (Matthes et al., 1995; Winberg et al., 1998). Therefore, it appears that normally the low level of dSema 2a on muscles is sufficient to repel inappropriate axon branches and confines synaptic arborization to specific muscles. This genetic analysis demonstrates dSema 2a can act selectively as a target-derived inhibitory synaptic arborization cue and a local repellent for extending motor neurons. Given the high levels of gSema 2a protein in muscle during motor neuron outgrowth, it will be interesting to investigate whether Sema 2a is involved in determining neuromuscular synaptic specificity in grasshopper. The chemorepulsive role for Sema 2a in growth cone pathfinding is similar to the functions described for the vertebrate secreted semaphorins, Collapsin-1 /Sema-III/D, which have also been shown to act as repulsive guidance cues, patterning sensory projections in the developing spinal cord and brain (Mark et al., 1997). Thus, despite the wide evolutionary divergence among the many species that express secreted semaphorins, there appears to be a high degree of conservation of semaphorin function. 97 Functional specificity within semaphorin domains In an effort to determine the structural elements necessary for Sema 2a function, we conducted function blocking experiments using antibodies generated against five different regions of Sema 2a. We found blocking the semaphorin domain had severe effects on the pathfinding of T i l growth cones, increasing axon pathfinding errors from approximately 10% in controls to 60% following perturbation of the central region of the sema domain. These defects were characterized by aberrant projections into areas of high expression of Sema 2a, indicating that the semaphorin domain is necessary for the chemorepulsive function of Sema 2a. Similarly, in vitro studies have demonstrated that the sema domain does confer functional specificity to secreted semaphorins. For example, the region of specificity within the sema domain of Coll-1 was narrowed to a 70 amino acid region which, when transplanted into the backbone of any other secreted semaphorin family member, was sufficient to determine the biological activity of the new chimeric molecule (Koppel et al., 1997). In Sema 2a this 70 amino acid stretch spans both fusion proteins SD1 and SD2, the N-terminal and central regions of the sema domain respectively, with all but the first amino acid lying within SD2 region. Interestingly, we found a significantly higher number of axonal malformations following perturbation of the central region of the sema domain compared to following perturbation of other regions of Sema 2a, suggesting this region is also of particular functional importance for invertebrate secreted semaphorins. If the semaphorin domain is responsible for the chemorepulsive activity of Sema 2a, what is the function of the C-terminal domain? In vitro studies have shown that members of the vertebrate secreted semaphorins need to be dimerized to be functional and that dimerization is dependent on cysteine residues in the C-terminus (Eickholt et al., 1997; Koppel et al., 1997; Klostermann et al., 1998; Koppel and Raper, 1998). In addition, it appears that secreted collapsin family members bind and activate their receptors as preformed dimers (Koppel and Raper, 1998). Therefore, the absence of axon abnormalities following perturbation with 98 antibodies directed against the last 172 amino acids of the C-terminus of Sema 2a may be a consequence of Sema 2a having been secreted as a covalent dimer. Alternatively, since neither gSema 2a and dSema 2a contain the carboxy-terminal basic domain characteristic of the vertebrate secreted semaphorins, a region proposed to be necessary for regulating the repulsive activity (Adams et al., 1997; Klostermann et al., 1998), the C-terminal portion of Sema 2a may not be required for chemorepulsion. In vitro studies using sema domain-Fc fusion proteins of vertebrate secreted semaphorins have also indicated that the C-terminus is not absolutely necessary for the repulsive activity; however, the activity of these chimeric proteins is considerably lower than full length Coll-1 (Eickholt et al., 1997; Koppel and Raper, 1998). In addition to promoting dimerization, the Ig and basic domains of vertebrate secreted semaphorins bind strongly to Neuropilin family members, components of the secreted semaphorin receptor complex (Feiner et al., 1997; He and Tessier-Lavigne, 1997; Giger et al., 1998). The partial attenuation of Sema 2a repulsion we observed following perturbation with antibodies directed against the entire C-terminal, including the Ig domain, may result from a disruption of receptor complex interactions. However, insect neuropilins have not been identified, therefore suggesting the secreted semaphorins may signal through a different mechanism in invertebrates. Nevertheless, it is possible that the conserved sema domain mediates the biological activity of secreted semaphorins, while the Ig and basic domains are involved in dimerizing and binding the ligand to the receptor complex and thereby potentiating its activity. Taken together, our in vivo function blocking data and the vertebrate studies indicate biological activity is domain specific and highly conserved among secreted semaphorins. Semaphorins act independently and in combination to guide growth cone pathfinding in vivo We have shown here that the secreted semaphorin Sema 2a acts as a repellent, however the nature of Sema l a signalling at the trochanter epithelial band remains unclear. While axon 99 defasciculation and additional branch formation following antibody perturbation is consistent with an inhibitory function of Sema la, the normal restriction of growth cone migration into the dorsal coxa, the adhesivity of T i l growth cones to the trochanter epithelium (Condic and Bentley, 1987a) and examination of Sema l a function on other neurons in the developing limb (Wong et al., 1997) supports an attractive/permissive function for Sema la. Although our experiments do not address this issue, it seems likely that the simple characterization of semaphorins as attractive or repulsive cues may not accurately reflect their function in vivo. In fact, it has become evident that single semaphorins may exert a wide range of functions, depending on the downstream signalling cascades stimulated in a neuronal growth cone. For example, Song et al. (1998) have recently shown that the vertebrate secreted semaphorin, Sema-III/D, can be switched from a repulsive guidance molecule to an attractive cue depending on the level of cGMP within the growth cone. The expression patterns of Sema 2a and Sema l a during the period of T i l pioneer outgrowth and throughout limb during development lead us to hypothesize that different members of the semaphorin family may act both independently and in combination to guide individual growth cones. Indeed, we observe that blocking the function of each semaphorin alone leads to spatially, temporally and phenotypically discrete axon malformations, demonstrating that these two semaphorins independently guide regions of T i l growth cone pathfinding. However, since the T i l pioneer growth cones contact regions of Sema 2a and Sema la as they approach the trochanter epithelium, there are also periods when the T i l growth cone must be receiving guidance information from both semaphorins. Consistent with this hypothesis, following simultaneous perturbation of both semaphorins we observed a significant increase in both frequency and range of axonal malformations. In these double blocking experiments single axon projections displayed abnormalities resembling hybrids of the individual blocking experiments, suggesting that not only do the two different semaphorin members provide discrete 100 guidance information to an individual growth cone, the growth cone must continuously integrate and compare this incoming information to pathfind correctly. In this study we provide direct evidence for an in vivo chemorepulsive gradient that is necessary for determining both the initial direction of axon outgrowth as well as subsequent growth cone pathfinding events. Furthermore, our in vivo results demonstrate that different classes of semaphorins can act discretely and simultaneously at the level of an individual growth cone, supporting the notion that it is the balance of their downstream signalling cascades which determines pathfinding decisions. 101 IV. GRADIENT SHAPE ENCODES GROWTH CONE GUIDANCE INFORMATION IN VIVO 102 Introduction Since Ramon Y Cajal first described the neuronal growth cone over a century ago, it has been proposed that spatial gradients of axon guidance molecules provide directional information to pathfinding growth cones. However, it was not until after Sperry published his chemoaffinity hypothesis in 1963, that extensive research was directed towards investigating the role of molecular gradients in growth cone guidance. Sperry postulated that neurons acquire unique molecular labels early in development and that topographic maps are, therefore, generated by matching molecules on the pre- and post-synaptic neurons. Although it is now recognized that additional mechanisms such as the activity-dependent fine-tuning of synapse formation also play a role in establishing neuronal connectivity, the basic tenet that selectivity of connections depends on the recognition of specific molecular cues in the vicinity of the target is widely accepted (reviewed by Goodman and Shatz, 1993). The reemergence of chemotropism as a mechanism for axon guidance catalyzed the effort to identify molecules that could guide growth cones from a distance. Many studies using the collagen-gel matrix assay, where explants of neuronal and target tissue are co-cultured in vitro within three dimensional collagen gels, have since demonstrated that growth cones can be guided by both diffusible chemoattractants and chemorepellents (e.g. Lumsden and Davies, 1983; Tessier-Lavigne et al., 1988; Fitzgerald et al., 1993; Pini, 1993; Serafini et al., 1994; Messersmith et al., 1995). Although the in vitro chemotropic studies co-evolved with the search for such molecular gradients in vivo, to date, only a few examples of gradient expression of molecules during periods of axon outgrowth have been documented (Norbeck et al., 1992; Seaveretal, 1996; Monschau et al., 1997; Braisted et al., 1997). Although considerable effort has been directed at characterizing chemotactic molecules and their receptors, the mechanisms that neuronal growth cones employ to detect these gradients remain largely unknown. For a growth cone to reliably pathfind up or down a gradient, a 103 difference in ligand concentration across the growth cone must be detectable above the background noise. Two possible mechanisms for growth cone detection of small changes in external gradients have been proposed and they differ on which aspect of the change in concentration across the growth cone spatial extent is most important, the absolute change or the fractional change (Walter et al., 1990; Goodhill and Baier, 1998; Goodhill, 1998). Distinguishing the mechanism that is employed by neuronal growth cones, however, has been limited by the scarcity of functional data on gradients in vivo. We have previously established that the secreted semaphorin, grasshopper Sema 2a, is a chemorepulsive guidance molecule expressed in the developing limb bud during the period of T i l pioneer neuron outgrowth (Isbister et al., 1999). In the present study, we demonstrate that Sema 2a is expressed within the developing grasshopper limb bud in perpendicular overlapping distal-proximal and dorsal-ventral gradients. Interestingly, these gradients are exponential in shape but differ in their steepness, and at stereotyped decision points it is the steepness of the gradient and not the absolute level of Sema 2a that provides the critical chemorepulsive information to the pathfinding T i l pioneer growth cones. Furthermore, we provide evidence that the T i l pioneer growth cone detects these gradients by measuring the fractional change in Sema 2a concentration across its spatial extent rather than detecting the absolute change. 104 Materials and Methods Immunocytochemistry For whole mount preparations, Schistocerca gregaria embryos at developmental stages 30.5% - 35% were dissected in phosphate buffered saline (PBS), fixed for 50 minutes in P E M -F A (0.1 M PIPES [pH 6.95], 2.0 m M EGTA, 1.0 m M M g S 0 4 , 3.7% formaldehyde), and washed for 1 hour with three changes in PBT (1XPBS, 0.5% Triton X-100, 0.2% bovine serum albumin) as previously described (Bentley et. al., 1979). The T i l pioneer cell bodies emerge from the anterior epithelium (Caudy and Bentley, 1986a) and, therefore, for limb fillet preparations, 30.5%-32.5% embryos were dissected and placed anterior side down on a poly-L-lysine coated cover slip (6 mg/ml). The exposed posterior epithelium of the T i l limb bud was cut lengthwise, and unrolled flat to reveal the pioneer pathway (O'Connor et al., 1990). A suction pipette was used to remove the mesodermal cells overlaying the limb epithelium. Limb fillets were then fixed as above for whole mount preparation. Following dissection and fixation, the embryos were incubated overnight at 4°C in primary antibody. For Sema 2a labeling, we used polyclonal antibodies generated against the conserved sema domain (SD1 and SD2; Isbister et al., 1999) and the purified IgGs were diluted 1:500 in PBT. We have previously established that all our independently generated Sema 2a antibody stocks detect the same single protein at the expected size for gSema 2a on western analysis and immunocytochemistry shows all the antibody stocks stain the identical pattern for protein expression (Isbister et al., 1999). In addition, the mRNA and protein are co-localized at all developmental stages examined, further confirming the specificity of our antibodies. For laminin labeling, purified IgGs were diluted 1:500 in PBT (IgG purified polyclonal antibodies generated against the y chain of grasshopper laminin were kindly provided by J. Bonner); for neuronal staining, goat anti-HRP (Jackson Immunoresearch Lab) was diluted 1:1000 in PBT. Following overnight incubation, the embryos were washed for 1 hr in PBT with three changes, 105 and incubated in the appropriate secondary antibody (1:250 dilution in PBT) for 2 hours at room temperature. Embryos were then washed for 1 hour with 6 changes of PBT. Fluorescently labeled embryos were mounted in Slowfade antifade reagent (Molecular Probes) and viewed under fluorescence microscopy. For Sema 2a and neuronal double labeling, embryos were simultaneously incubated in the two primary antibodies overnight at 4°C (1:500 rabbit anti-Sema 2a, 1:1000 goat anti-HRP diluted in PBT). The embryos were then washed for 1 hr in PBT with three changes, and incubated for 2 hours at room temperature in PBT containing the appropriate secondary antibody (1:250 FITC conjugated donkey anti-rabbit, 1:250 Texas Red conjugated donkey anti-goat). The embryos were washed and mounted as described above. Image acquisition. For wide field images, fluorescent labeled fillet and whole mount limbs were illuminated with a Nikon 100 W halogen light with the appropriate filter set (Chroma Tech), and shuttered with a computer-controlled Lambda 10-2 shutter (Sutter Instrument Co.). Limbs were imaged with a Princeton Instruments MicroMax CCD camera (Kodak chip K A F 1400) and digitized with MetaView Imaging System 3.6 (Universal Imaging Corp., PA). Confocal images were collected with a Bio-Rad M R C 600 system attached to a Zeiss Axioskop microscope and a 40 X 1.3 N A Zeiss objective or a 25 X 0.8 N A Zeiss objective (Laser intensity = 1-10%; confocal pinhole = 3 Bio-Rad units). Serial images along the z-axis (z-series) were obtained through the entire specimen (step size = 2 pm) and a maximal-intensity projection was used to generate a two-dimensional representation of maximum Sema 2a fluorescence. A maximal intensity projection flattens 3-D images by creating an image of the maximal pixel value for each pixel across the sections. 106 Gradient analysis. To quantify Sema 2a and laminin protein expression, 15-100 um wide linescans were taken along both the distal-proximal and dorsal-ventral axis (MetaView Imaging System 3.6, Universal Imaging Corp., PA). The distal-proximal linescan started at the distal tip of the flattened anterior compartment epithelium and included the region of limb epithelium where the T i l cell bodies were located. The dorsal-ventral linescan started at the dorsal edge of the flattened epithelium and spanned the trochanter limb segment. Since the process of slicing the posterior surface of the limb bud during fillet preparation often led to the removal of the majority of the posterior epithelium, we scanned only the anterior compartment along the distal-proximal and dorsal-ventral axes. Some limbs were filleted such that the posterior epithelium was largely intact, in these limbs the expression of Sema 2a was observed to decrease from dorsal to ventral along both the anterior and posterior epithelial compartments. For each linescan, the fluorescence intensity profile and corresponding limb position were exported into a spreadsheet for analysis. Each fluorescence intensity profile was scaled to its maximum intensity and the relative intensities were plotted against limb position. Relative intensity profiles along the dorsal-ventral axis were scaled to the maximum intensity within the limb, which invariably occurred within the distal-proximal profile. The position of the T i l cell body along the distal-proximal axis was also recorded for each trace. The dorsal-ventral profiles were combined and averaged by aligning to the dorsal edge of the limb, while the distal-proximal intensity profiles were combined and averaged by aligning to the distal limb tip. In addition, we averaged the distal-proximal intensity profiles by aligning to the T i l cell bodies; however, the resulting intensity profile and decay constants were not significantly different from the averaged profile aligned to the distal tip (data not shown). Averaged curves were fit with either linear regression or single exponential decay using P C L A M P (Axon Instruments Inc.) and Microcal Origin software. A linear regression fit determined the slope, AF; that is, the change in fluorescence per pm. A single exponential decay 107 fit determined the decay rate, x; that is, the distance in pm over which the intensity decayed by 63%. For statistical analysis, curve fits were performed on the individual distal-proximal and dorsal-ventral plots and two-tailed unpaired t-tests were used to compare slopes or decay rates. Antibody blocking experiments IgGs and Fab fragments were diluted into freshly made supplemented RPMI culture media as previously described (polyclonal antibody stock SD2; Isbister et al., 1999). Prior to culturing, embryos from a single clutch were sterilized, dissected and the entire amnion and dorsal membrane removed from the embryo to ensure access of the antibodies during culturing. As embryos within a single clutch typically differ by less than 1% of embryonic development (Bentley et al., 1979), each clutch was randomly divided into experimental and control groups, with several embryos fixed immediately to determine developmental stage at the start of culturing. Embryo culturing began at 30% of development, just prior to T i l pioneer neuron axonogenesis. Following approximately 16 hours in culture, embryos were fixed and immunostained with antibodies to HRP (Jan and Jan, 1982) for visualization of T i l axons and other neurons. For each culture experiment, limbs from IgG and Fab cultured embryos were compared with limbs from control cultured embryos (media alone and preimmune-antibody). The data are presented as a percentage of the abnormal T i l pathways observed. For both cultured and untreated embryos, the T i l pathway was scored as abnormal for one or more of the following observed characteristics: reorientation and distal projection of the proximal T i l cell body axon; direct projection of the distal T i l cell body axon into the distal limb bud tip; dorsal projection of one or both axons for more than 50pm; and failure to initiate a single axon, typically characterized by multiple short, randomly oriented projections from the cell body. 108 Results Sema 2a is expressed in overlapping perpendicular distal-proximal and dorsal-ventral gradients within the developing limb bud. In the grasshopper limb bud the pair of T i l pioneer neurons at the limb-bud tip are the first neurons to establish a projection to the CNS. The projection is extremely stereotyped in both the substrates contacted and the steering decisions made by the growth cones as they migrate (Fig. 19^ 4; reviewed by Bentley and O'Connor, 1992). The well-characterized nature of this projection, combined with the large size of the T i l pioneer growth cones, provides a powerful model system for investigating growth cone pathfinding mechanisms in vivo. The initial characterization of Sema 2a indicated that during the period of Ti 1 pioneer axon outgrowth and pathfinding, this molecule is highly expressed in the distal and dorsal limb compartments (Fig. 195) and functions to repel axons from extending into these inappropriate regions (Isbister et al., 1999). Interestingly, the Sema 2a expression appeared to decrease in a gradient fashion and it was therefore hypothesized that the T i l growth cones migrate down this chemorepulsive gradient towards the CNS. The three dimensional nature of the whole mount limb preparation may, however, confound interpretation of the true expression pattern of Sema 2a. For example, areas of perceived high expression could result from viewing overlapping layers of epithelium, regions of thicker epithelium, or the additive effect of Sema 2a positive mesodermal cells within the lumen of the developing limb bud. Therefore, to establish whether Sema 2a protein is indeed expressed in a gradient during T i l axonogenesis, we stained developing limb bud fillets at the stage of T i l pioneer outgrowth (Fig. 2(L4). Filleting the limbs enabled the epithelium to be unrolled and flattened into a sheet and, with the subsequent removal of the overlying mesodermal cells, allowed for an unobstructed view of the epithelial expression of Sema 2a. 109 Figure 19. Sema 2a protein expression during the Til pioneer projection into the CNS. A , Schematic of the T i l pioneer neuron pathway at -36% embryonic development. The pair of sibling T i l pioneer neurons arises from the underlying epithelium between 29-30% of embryonic development. At approximately 30.5% of development the T i l growth cones emerge from their cell bodies and begin to extend axons proximally along the limb axis towards the CNS. Once contact with the Tr cell has been made, usually around 33% of development, the T i l growth cones reorient to extend ventrally along the trochanter epithelium. At about 34% development the T i l growth cones reorient proximally and exit the trochanter to contact the C x i guidepost cells. By 36% the T i l pioneer growth cones have extended proximally from the C x i cells into the CNS. B, Sema 2a and anti-HRP double staining at 32% of embryonic development (green: Sema 2a protein; red: HRP-stained neurons; arrow demarcates T i l growth cones; arrowheads demarcate the region of the trochanter limb segment). Scale bar, 40pm. 110 Figure 20. Sema 2a gradient analysis. A , A representative wide field image of a Sema 2a labeled limb fillet at -31% embryonic development. T i l pioneer growth cones are shown migrating towards the trochanter (arrow), (green: Sema 2a protein; red: HRP-stained neurons; dashed line denotes proximal boundary of filleted limb and beginning of unfilleted abdomen). B, A pseudocoloured image of fillet shown in (A) illustrating the orientation of the distal-proximal and dorsal-ventral linescans. C, A representative wide field image of a Sema 2a labeled limb fillet (green: Sema 2a protein; red: HRP-stained neurons). In this fillet the proximal T i l cell body is extending its growth cone proximally; whereas, the growth cone of the distal T i l cell body has emerged from the distal pole and is in the process of reorienting ventrally (arrow). D, A confocal maximum intensity projection of the fillet shown in (C). E, Relative intensity profile of Sema 2a protein expression along the distal-proximal limb axis. The wide field and confocal traces are equivalent, showing maximum protein expression in the distal tip of the limb bud and decreasing proximally along the limb axis. F, Relative intensity profile of Sema 2a expression along the dorsal-ventral limb axis, showing maximum expression in the dorsal compartment and decreasing ventrally across the limb. The maximum protein expression observed in the dorsal-ventral axis is -65% of the maximum observed along the distal-proximal axis. Scale bar, 50pm. 112 dist. Distance (um) prox. dors. Distance (um) vent. 113 To determine the relative levels of Sema 2a protein expression within the developing limb bud, the intensity of Sema 2a immunofluorescence was measured using linescans oriented along the distal-proximal and dorsal-ventral axis of each limb fillet (Fig. 205). To control for possible variability in thickness and multiple focal planes over the epithelial sheet, we confirmed our wide field limb fillet results in optical sections of uniform thickness using confocal microscopy (Fig. 20C, D). The relative fluorescence intensities were plotted against their respective position in the limb and the resulting linescan profile of the Sema 2a immunofluorescence indicated that protein expression decreases steadily along both limb axes, with the maximum fluorescence occurring in the distal and dorsal regions (Fig. 20E, F). The wide field image and the maximum intensity projection of the confocal image of the same limb fillet produced equivalent linescan traces, confirming that Sema 2a protein expression decreased along both the distal-proximal and dorsal-ventral limb axes. The average of 24 limb fillets revealed that the pattern of Sema 2a protein expression along the distal-proximal axis is highly consistent across limb buds. The highest expression occurred in the distal tip and decreased towards the proximal limb segment (Fig. 21^4). The averaged profile along the dorsal-ventral axis also revealed a highly stereotyped pattern of Sema 2a protein expression. The greatest expression occurred in the dorsal limb and decreased towards the ventral compartment (Fig. 215). These results demonstrate that during the period of T i l pioneer axonogenesis and pathfinding, Sema 2a protein is expressed in a distal-proximal and a dorsal-ventral gradient. The two gradients are perpendicular to each other, most obviously overlapping in the femur. The distal tip of the limb bud (tarsus and tibia) is characterized predominantly by the distal-proximal gradient of Sema 2a, while the dorsal-ventral gradient of Sema 2a is dominant in the proximal femur and trochanter. 114 Figure 21. Sema 2a is expressed in exponential distal-proximal and dorsal-ventral gradients. A , The average distal-proximal Sema 2a gradient (dark lines; gray lines show ±SEM) reveals a highly consistent profile with peak protein expression observed in the distal tip and steadily decreasing proximally. The T i l cell bodies are located 56 - 112pm proximally from the distal limb tip as indicated. The average maximum Sema 2a expression level that was observed along the dorsal-ventral axis was 79% of that observed along the distal-proximal gradient (arrow). Inset: Curve fit analysis revealed the distal-proximal gradient of Sema 2a is comprised of a steep exponential gradient (decay constant, T = 20pm) starting at the distal tip and extending to the edge of the T i l cell body range, followed by a linear gradient decaying towards the trochanter. B, The average relative intensity profile of the dorsal-ventral Sema 2a gradient shows dorsal peak protein expression, steadily decreasing towards the ventral limb compartment. Inset: the dorsal-ventral gradient was best described by a single exponential with a decay constant (T) of 112pm. Scale bar for insets; y-axis is 0.20 units relative intensity, x-axis is 50pm. 115 a: 2 \ • • • 0 50 100 150 200 250 300 distal Distance (Lim) proximal 1 1 6 Sema 2a gradient expression is highest and steepest distal to the Til pioneer cell bodies The profile of the averaged distal-proximal Sema 2a immunofluorescence plot indicated that the gradient may be comprised of two distinct components: a steep gradient distal to the T i l cell bodies, and a second more shallow gradient proximal to the cell bodies (Fig. 2 \ A ) . Curve fit analysis demonstrated that the distal-proximal plot is indeed best described by two separate equations, an exponential decay for the region from the distal limb tip to the cell bodies and a linear decay for the region proximal to the cell bodies (Fig. 21,4 insets). This proximal gradient did not fit an exponential, however it is conceivable that the distal-proximal gradient may actually continue to decay exponentially proximal to the cell bodies, however the profile may become distorted as the overlapping contribution of the dorsal-ventral gradient becomes more apparent. To confirm that the region distal to the cell bodies is best described by an exponential equation, we fit equations from the distal tip to the distal edge of the cell body range, 56pm, and to the proximal edge of the cell body range, 112pm. As would be predicted for an exponential curve, there was no difference between the taus for the two plots (To-56um = 20; In.] i2u.m = 20). However, fitting linear equations to the data gave radically differing slopes depending on the sections of the curve selected (e.g. AFn-56um = -0.57pm"1; AFo-H2u.m = -0.21pm"1), thus further verifying that the distal-proximal gradient in the region distal to the T i l cell bodies is exponential. Curve fit analysis showed that the immunofluorescence intensity plot of the average dorsal-ventral Sema 2a gradient is best described by a single exponential curve with a decay constant (x) of 112pm (Fig. 215, inset). Interestingly, the exponential component of the distal-proximal Sema 2a gradient is considerably steeper than the exponential gradient along the dorsal-ventral axis (x = 20pm versus x = 112pm; rtest of individual profiles, p<0.05). In addition to a steeper gradient of Sema 2a protein in the distal limb segment, the levels of Sema 2a protein expression in this region are 117 higher than observed elsewhere in the limb. On average, over the most distal ~20p.m of the limb tip, Sema 2a expression sharply rises to levels 20% above the maximum levels observed in the dorsal limb compartment (arrow at .79 relative intensity on distal-proximal profile designates the maximum intensity observed along dorsal-ventral axis, Fig 21^4). Therefore, in the ~30pm region immediately distal to the Ti 1 cell bodies the levels of Sema 2a protein are actually comparable to those observed in the dorsal regions of the dorsal-ventral gradient, even though the gradient is rising more steeply. To verify that the immunofluorescence and linescan analysis accurately reflect protein expression patterns we stained developing limb bud fillets at the stage of T i l pioneer outgrowth for grasshopper laminin, a uniformly expressed basal lamina protein (Bonner and O'Connor, Society for Developmental Biology Abstract, 1999). The averaged distal-proximal and dorsal-ventral laminin immunofluorescence plots display slopes around zero. In addition, the absence of an average value of 100% reveals the randomness of the position of maximum fluorescence among the individual fillets (Fig. 22/4, B). Steepness of the gradient, not absolute levels ofprotein expression, provides the critical chemorepulsive guidance information. Prior to axonogenesis, the T i l pioneer neurons extend multiple filopodia in all directions around their cell body perimeter. After sampling of the environment by these initial filopodia, the proximal T i l cell body will typically project its growth cone and nascent axon from the proximal pole; whereas the distal T i l cell body may initiate its axon either near its proximal or distal pole (Lefcort and Bentley, 1989). During the early stages of axonogenesis, the T i l growth cones interact predominantly with the steep distal-proximal gradient of Sema 2a, and the first decision the T i l growth cone must make is to migrate proximally down the gradient of Sema 2a. By 33% of development the growth cones have typically contacted the Tr guidepost cell within the trochanter epithelium, at this position they are primarily interacting with the shallow dorsal-118 Figure 22. Laminin protein expression within the grasshopper limb bud at 31% embryonic development. A , B, The average relative intensity profiles for laminin protein expression (dark lines; gray lines +SEM), illustrating uniform expression along both the distal-proximal and dorsal-ventral limb axes. The profiles were linear with a slope (AF) near zero and no region of peak expression. 119 A 55 c CD 03 B CO 0 a: AF = -.010|im"1 0 50 distal 100 150 200 250 Distance (|um) proximal .6 AF = .015|nm"1 0 50 dorsal 100 150 200 250 Distance (jam) ventral 120 ventral gradient of Sema 2a. At this stage, the growth cone makes a second guidance decision and reorients to migrate ventrally within the trochanter, again down the gradient of Sema 2a. Therefore, at two critical decision points in the T i l pioneer pathway, the T i l growth cone must decide to migrate up or down a gradient of Sema 2a. These findings raised the question of whether guidance information is encoded in the steepness of the gradient, or alternatively, whether Sema 2a conveys the degree of chemorepulsion based on the absolute level of Sema 2a expression. To determine if the steepness of the Sema 2a gradient has functional significance for the pathfinding of Ti 1 pioneer growth cones, we recorded the type and frequency of errors that occur during normal T i l growth cone pathfinding towards the CNS (30-35% development). In normal developing grasshopper embryos, approximately 10% of the T i l pathways exhibit pathfinding errors. Analysis of untreated embryos at -36% development revealed that the majority of T i l pioneer projection errors occur during growth cone interaction with the shallow dorsal-ventral Sema 2a gradient, typified by axons projecting aberrantly into the dorsal compartment of the limb within or near the trochanter (Fig. 235, Fig. 244). Rarely did we observe errors occur during growth cone interaction with steep distal-proximal gradient of Sema 2a, nevertheless, when errors did occur within this region they were typically either direct extension of the distal T i l cell body axon into the distal tip of the limb bud or failure to initiate a single axon (Fig. 23C, D; Fig. 244). Therefore, errors within both gradients were typified by aberrant projections up the gradient into regions of higher Sema 2a expression and far fewer projection errors within the steep distal-proximal gradient of Sema 2a compared to the shallow dorsal-ventral gradient. This skewed distribution of aberrant phenotypes suggests that the steep distal-proximal gradient of Sema 2a confers a greater degree of chemorepulsion to the pathfinding T i l growth cones. Similarly, a recent in vitro study has demonstrated that temporal retinal ganglion axons will extend up steep gradients of repellents and, furthermore, the maximum distance reached up a given gradient decreases with increasing slope (Rosentreter et al., 1998). Although this study 121 Figure 23. Til pioneer growth cones occasionally misproject up the chemorepulsive gradients of Sema 2a. A, A representative example of the T i l pioneer projection into the CNS. B , A representative example of the dorsal projection error phenotype. The arrow denotes the misguided T i l pioneer growth cone migrating within the dorsal trochanter; the sibling T i l growth cone has successfully completed its projection into the CNS. C, A representative example of the distal projection error phenotype, these errors were typically direct projection of the distal T i l cell body axon into the extreme distal tip of the limb bud. Arrows denote proximal and distal projecting growth cones of the sibling T i l cell bodies. D, A representative example of the failure to initiate a single axon phenotype; typically, the T i l pioneer cell bodies displayed multiple short axons projecting radially from around the cell body periphery. Arrowheads denote the trochanter limb segment. Scale bar, 60pm in (A) and (B), 50pm in (C) and (D). 122 normal ' IB dorsal multiple short axons ^ 123 Figure 24. Steep distal-proximal gradient of Sema 2a ensures Til pioneer axonogenesis and proximal outgrowth. A , Summary of the phenotypes and frequency of T i l axon projection errors in normal, untreated embryos. By 36% embryonic development, when the T i l growth cones should have completed their projection into the CNS, -10% of the T i l projections exhibit pathfinding abnormalities (dashed fill). The majority of aberrant projections are axons misguided into the dorsal limb compartment within and near the trochanter (black fill). Few aberrant projections are observed within the distal limb segment, (comprising both distal projecting errors and failures to initiate a single axon; gray and white fill , respectively). Data are pooled from 8 experiments. B, Summary of the phenotypes and frequency of T i l axon projection errors in normal, untreated embryos at early stages of T i l pioneer axon outgrowth (-31.5%). We observe - 7 % of the T i l projections are aberrant, again with the majority of misguided axons observed within the dorsal limb compartment. Data are pooled from 4 experiments. C, Summary of the phenotypes and frequency of T i l axon projection errors following functional perturbation of the Sema 2a gradients. RPMI and pre-immune ( lpM) cultured embryos exhibit the same error profile as the untreated embryos, with the majority of axon pathfinding errors occurring within the dorsal limb compartment within and near the trochanter. Embryos cultured in the presence of function blocking antibodies ( lpM) directed against Sema 2a during the period of T i l pioneer axonogenesis and outgrowth exhibit a -10-fold increase in T i l pioneer projection abnormalities. Following functional perturbation of the Sema 2a gradients, misguided axons into the distal limb bud and T i l cell bodies failing to initiate a single axon occur as frequently as projection errors into the dorsal limb compartment (p = 0.20). Data are pooled from 5 experiments. For all panels, n = number of T i l projections scored. Error bars: +SEM. rtests were performed to compare the frequency of errors within the dorsal-ventral gradient (dorsal errors; black fill) to the 124 frequency of errors within the steep distal-proximal gradient (combined distal and multiple short axons errors; gray and white fil l , respectively); * p < 0.05. 125 A 16 CO | 14 *-t—« F 8 -i—» 6 | 4 H ro « 0 C 70 i g 60 o "S 50 o. 40 - 30 ro S 20 .Q ro 10 0 B 16 i a) c o c 14 o i o 110 c ro CD ro 8 6 4 2 H 0 36% development 31.5% development TOTAL ERRORS dorsal errors distal errors | multiple short axons control culture Sema 2a block 126 does not address the role of gradients in determining growth cone pathfinding decisions, it does indicate that axon extension is not dependent on the absolute concentration of repellent within the gradient. Therefore, the greater degree of chemorepulsion in the distal-proximal gradient of Sema 2a, evident by the fewer projection errors up this gradient, could be conveyed by the steepness of the gradient or, alternatively, by the absolute level of Sema 2a protein. However, considering that the absolute level of Sema 2a in the dorsal trochanter epithelium is similar to that observed just distal to the T i l cell bodies, yet the dorsal trochanter is a region the T i l growth cone will extensively sample prior to committing to a ventral turn and more frequently extend erroneous axons into (Caudy and Bentley, 1986a; O'Connor et al., 1990; Isbister and O'Connor, 1999; Isbister et al., 1999; this study), we favour gradient steepness as the critical parameter conveying the degree of chemorepulsion to the pathfinding T i l growth cone. In order to confirm that it is the gradient steepness that confers the critical guidance information to the pathfinding T i l growth cones, and not the absolute level of Sema 2a, we asked whether at early stages of axonogenesis the T i l pioneer axons transiently extend up the steep distal gradient and are subsequently repelled by the high absolute levels of Sema 2a protein in the extreme distal tip. Given that the levels of Sema 2a just distal to the T i l cell bodies are similar to the levels in the dorsal limb compartment, where the T i l axons often project erroneously, it would be expected that a similar frequency of projections into the distal tip should be observed at these earlier stages of development. Analysis of axon projection errors at earlier stages of T i l outgrowth (30.5 - 31.5%), however, did not reveal an increase in frequency of T i l axon aberrant projections into the distal tip (Fig. 245). A skewed distribution of error subtypes is evident even at these earlier stages, with extensions into the dorsal limb compartment comprising the majority of axon misprojections. These results indicate that it is the steepness of the distal-proximal Sema 2a gradient that conveys the higher degree of chemorepulsion to the pathfinding T i l growth cone, thus minimizing projection errors. 127 We also observed that distal T i l cell body growth cones that emerge from the distal pole will often extend up the steep repellent distal-proximal gradient of Sema 2a, yet these growth cones typically reorient immediately to extend proximally and fasciculate with the proximal T i l cell body axon. Interestingly, we found that these distal projecting T i l growth cones reoriented by first turning ventrally, possibly detecting the dorsal-ventral repellant gradient of Sema 2a (Fig. 20C, Fig. 25). Therefore, similar to temporal retinal ganglion axons, the T i l pioneer growth cones will extend up both steep and shallow repellent gradients of Sema 2a, but typically extend further within the shallow gradient. Furthermore, the distal projecting T i l pioneer growth cones reorient in the region where the steepness of the Sema 2a gradient differs but the absolute expression levels are similar, again confirming that the steepness of the Sema 2a gradient encodes the critical chemorepulsive guidance information. To further examine whether it is the gradient of Sema 2a which initiates and maintains the proximal projection of the T i l pioneer axons, we performed antibody blocking experiments during the period of T i l pioneer neuron axonogenesis and outgrowth. Blocking Sema 2a function leads to a 10-fold increase in T i l pioneer projection abnormalities with distal projecting axons and T i l cell bodies with multiple short axons occurring as frequently as dorsal projecting axons (Fig. 24C; rtest, p = 0.20). The disappearance of the skewed distribution of error subtypes when the function of Sema 2a is blocked further supports that the steep distal gradient of Sema 2a is responsible for maintaining the low frequency of projection errors within the distal limb segment. Surprisingly, following perturbation of Sema 2a function we often observed the proximal T i l cell body extending its axon into the distal segment. This distal reorientation of the proximal T i l axon suggests that the steep distal-proximal gradient of Sema 2a may oppose either an attractive cue emanating from the distal tip or, alternatively, a proximal-distal oriented repulsive cue. Furthermore, in addition to growth cone guidance, the steep distal-proximal chemorepulsive gradient of Sema 2a appears to play a role in determining the initial site of T i l 128 Figure 25. Growth cones emerging from the distal pole of the distal Til cell body reorient immediately to migrate down the Sema 2a gradients. The proximal T i l cell body initiates its growth cone from the proximal pole and continues to migrate down the distal-proximal gradient of Sema 2a. The distal T i l cell body, however, can initiate its growth cone from the distal pole (arrow). This growth cone has already turned ventrally and is now in the process of reorienting proximally to extend towards, and fasciculate with, the proximally migrating sibling T i l axon. Arrowheads demarcate trochanter limb segment. Scale bar, 20pm. 129 130 growth cone emergence and the subsequent establishment of a single axon, as revealed by the increased failure to initiate a single axon following Sema 2a function perturbation. Absolute levels of Sema 2a may constrain growth cone size. An alternative explanation for the increased chemorepulsion conferred by the distal-proximal gradient of Sema 2a is that the growth cones migrating within this gradient are exposed to more total repellent. For example, although the levels of Sema 2a surrounding the cell bodies are similar to the levels in the dorsal-most region of the trochanter, perhaps only a fraction of the filopodia are interacting with the dorsal region; whereas during migration within the steep distal-proximal gradient, the entire growth cone surface is surrounded by these high levels of Sema 2a. This would support the argument that the absolute level of repellent is responsible for the decreased frequency of error into the distal limb compartment. To test this hypothesis, we calculated the total amount of Sema 2a encountered by the T i l growth cone at pathfinding decision points within the two gradients. The first decision point occurs when the growth cone emerges from its cell body, as it must decide to migrate down the steep distal-proximal gradient of Sema 2a. The second major reorientation occurs within the shallow dorsal-ventral gradient of Sema 2a at the trochanter, at this decision point the T i l growth cones reorient to migrate ventrally again down the chemorepulsive gradient of Sema 2a. To calculate the area of limb epithelium sampled by a typical T i l growth cone, we measured the average growth cone length and width at these two points. We determined that when a growth cone first emerges from its cell body, it will typically extend 35 ± 4pm along the distal-proximal gradient and displays a width of 31 ± 4pm; whereas, within the trochanter the typical growth cone extends 81 ± 6pm along the dorsal-ventral gradient and displays a depth of 26 ± 1pm (n = 12 and n = 18, respectively). The total amount of Sema 2a encountered by the T i l growth cone was then calculated by applying these growth cone parameters to the distal-proximal and dorsal-131 ventral averaged relative intensity curves for Sema 2a. We found that although the T i l growth cone increases in size to sample twice the area of limb epithelium within the trochanter as the mid-femur (2106pm2 versus 1085pm2, respectively), this increase in growth cone size is proportional to the decreasing distal-proximal gradient of Sema 2a. Consequently, the total amount of Sema 2a within the region occupied by the growth cone is similar during interaction with the steep distal-proximal and shallow dorsal-ventral gradient (224034±9803Rel.Int. versus 247918±16033Rel.Int.; rtest, p > .05). The measurement of area calculated by length and width represents the region spanned by the growth cone as a square, however, this measurement may not be an accurate indication of the area sampled by the growth cone. Therefore, a second calculation was performed using previously published values for T i l growth cone sampling area that more closely resembled growth cone morphology (O'Connor et al., 1990), however the results were similar (92938±4067Rel.Int. for femur and 101265±6549Rel.Int. for trochanter; rtest, p > .05). Thus, further verifying that the absolute level of Sema 2a detected by the pathfinding Ti 1 growth cone does not confer the critical chemorepulsive guidance information. As the T i l growth cone migrates along the distal-proximal axis towards the trochanter, the decrease in Sema 2a expression is correlated with an increase in growth cone size. The changes in morphology, therefore, could result from a decrease in surround repulsion, which enables the growth cone to expand and branch until the total amount of Sema, 2a encountered reaches a critical level that inhibits growth cone size. These results indicate that the morphology of the T i l growth cone may be constrained by the absolute levels of Sema 2a. Til growth cones detect gradient shape using the fractional change mechanism. How does the T i l growth cone detect gradient steepness? Two possible mechanisms that a growth cone could employ to detect and internally amplify small changes in concentration across its spatial extent have been proposed: (1) the absolute change model, and (2) the fractional change model (reviewed by Goodhill, 1998; Goodhill and Baier, 1998). The absolute 132 change model proposes that the growth cone detects an absolute change in ligand concentration (AC) across its spatial extent (Fig. 26^4). Whereas, gradient detection by the fractional change mechanism is achieved by the growth cone measuring the change in concentration across its spatial extent as a fraction of the maximum concentration detected. In this model the growth cone sets the maximum concentration of ligand to 100% (C), where 0% is at the baseline concentration for the gradient, and the change in ligand concentration across the growth cone is measured as a fraction of this 100% maximum concentration, AC/C (Fig. 26^4). The optimal gradient shape for detection by these two mechanisms differs (reviewed by Goodhill, 1998; Goodhill and Baier, 1998). The absolute change mechanism best detects linear gradients, where the change in concentration is constant at all positions along the gradient, as represented by a slope. The fractional change mechanism, however, best detects exponential gradients where the fractional change in concentration is constant anywhere along the gradient, as represented by a tau (the distance along the gradient it takes to lose 63% of the initial concentration). The transition from an exponential gradient to a linear gradient along the distal-proximal axis suggests that the growth cone changes its gradient reading mechanism from fractional to absolute. Alternatively, the linear decay profile may reflect the overlapping contributions of the two perpendicular exponential gradients and, therefore, the growth cone may continue to employ the fractional change mechanism. We predict that the T i l growth cone employs the fractional change mechanism to determine the change in Sema 2a concentration across its spatial extent, based on the exponential shape of the Sema 2a gradient in the region of the two stereotyped decision points. Given that there are fewer erroneous projections up the steep distal-proximal gradient, it follows that for the steepness of the gradient to confer the degree of chemorepulsion, the mechanism of gradient detection employed by the T i l growth cone must be capable of detecting the difference in steepness between the two gradients. For example, i f the T i l growth cone uses the fractional change mechanism to detect the steepness of the gradient, as the shape of the 133 Figure 26. Til pioneer growth cones use the fractional change mechanism of gradient-reading to detect the steepness of the exponential gradients of Sema 2a. A , Schematic illustrating the calculation of change in gradient across a growth cone using the absolute and fractional change mechanisms of gradient reading. AC represents the absolute change in ligand concentration across the growth cone. AC/C calculates the fractional change in ligand concentration across the growth cone. C represents the maximum ligand concentration detected at the one edge of the growth cone; this value for C is set to 100%, where 0% is the baseline concentration for the gradient. AD represents the distance the growth cone spans along the gradient. AD varies during development since the T i l growth cone expands with proximal migration towards the trochanter. Alternatively, the fractional change in concentration across the growth cone can be calculated using the formal equation: l - e ^ D / T \ where T represents the single exponential decay constant. B, At 31.5% of development, the young T i l growth cone spans an average distance of 35 pm along the distal-proximal gradient of Sema 2a. Depicted is a typical distally emerging growth cone from the distal T i l cell body to illustrate its size and position along the gradient at 31.5%). The absolute change (AC) in Sema 2a across the growth cone is 0.11 Rei.Int.; however, the fractional change (AC/C) in Sema 2a is 0.1 lRel.Int./0.14Rel.Int.=81%. C, By 33% of development the T i l growth cone has contacted the Tr guidepost cell within the trochanter and has increased in size to span an average distance of 81 pm along the dorsal-ventral gradient of Sema 2a (as indicated by the growth cone; correct size and position). Within the trochanter, the absolute change (AC) in Sema 2a across the growth cone is 0.12Rel.Int, while the fractional change (AC/C) is 0.12Rel.Int./0.23Rel.Int.=51%. D, Table comparing absolute change in Sema 2a expression and relative change in Sema 2a expression across the typical T i l growth cone at the two limb positions. The absolute change is similar; however, the fractional change across the growth cone is markedly different at the two 134 positions. The fractional change mechanism detects a 51% change in Sema 2a concentration across the growth cone within the trochanter, a concentration change significantly less than the 81% detected by the same mechanism during growth cone interaction with the steep distal-proximal gradient (rtest, p < 0.05). The absolute change mechanism detects a change in concentration across the T i l growth cone within the trochanter of 0.12F, a value equivalent to that detected across the growth cone within the distal-proximal gradient (rtest, p = 0.15). 1 distal-proximal gradient 0.14 35 urn C .8 r CD CD .2 i D dorsal-ventral gradient 50 100 150 distance 50 100 150 distance 200 250 dist-prox dors-vent p Absolute Change AC Fractional Change AC / C 0.11 F 0.12 F 0.15 81 % 51 % < 05 136 gradient predicts, the fractional change across a growth cone within the exponential region of the distal-proximal gradient should be significantly higher than the fractional change across a growth cone interacting with the dorsal-ventral gradient within the trochanter. The higher fractional change across the growth cone would signal more repulsion and, consequently, result in fewer erroneous growth cone projections up the distal-proximal chemorepulsive gradient. Therefore, to test which gradient-reading mechanism is likely employed by the Ti 1 growth cone, we used both the absolute and fractional change mechanisms to calculate the average change in Sema 2a concentration across the growth cone during interaction with the two gradients. To calculate change in Sema 2a concentration across growth cones interacting with each of the exponential gradients, we applied the distance a typical T i l growth cone extends along the distal-proximal and dorsal-ventral gradient (35 + 4pm and 81 + 6pm, respectively) to the averaged Sema 2a intensity profiles. We found that using the fractional change mechanism of gradient-reading the T i l growth cone would detect an 81% and a 51% drop in Sema 2a concentration across its spatial extent during interaction with the exponential distal-proximal and dorsal-ventral gradient, respectively (Fig. 26B, Q . Analysis of the individual plots confirmed that the fractional change across T i l growth cones within the exponential distal-proximal Sema 2a gradient was significantly greater than the fractional change of Sema 2a across growth cones within the dorsal-ventral gradient of Sema 2a (rtest, p < 0.05). These results demonstrate that the fractional change mechanism of gradient reading is indeed sensitive enough to detect the difference in gradient steepness. In contrast, there was no significant difference in the absolute change of Sema 2a across T i l growth cones within the two gradients (Fig. 26B, C; rtest, p = 0.15). Our observations are consistent with the present theoretical models of growth cone gradient detection which calculate that the fractional change mechanism is most sensitive for the detection of exponential gradients while the absolute change mechanism is most sensitive for linear gradients (Goodhill, 1998; Goodhill and Baier, 1998). 137 Our calculations were determined for a typical growth cone emerging from the distal pole of the distal T i l cell body. We also applied these calculations to a typical growth cone emerging from the proximal T i l cell body. For the proximal T i l cell body growth cone the fractional change mechanism would still detect an 81% change in concentration, as would be expected since fractional change is constant anywhere along an exponential curve. However, the absolute change mechanism would detect only a small concentration change (-0.002F; edge of proximal T i l cell body is ~112pm from the distal tip). Therefore, the absolute change mechanism of gradient detection would predict that the distal-proximal gradient actually provides less chemorepulsion to the proximal T i l growth cone than the shallower dorsal-ventral gradient (0.002F versus 0.12F, respectively). Less chemorepulsion would result in more frequent erroneous axon projections up the distal-proximal gradient in comparison to the dorsal-ventral gradient, a hypothesis directly contrary to the data. We conclude, therefore, that the T i l growth cones are not likely to use the absolute change mechanism of gradient reading to detect the difference in steepness between the distal-proximal and dorsal-ventral Sema 2a gradients. Taken together, the difference in the steepness between the distal-proximal and dorsal-ventral Sema 2a gradients, the similarity in the absolute levels of Sema 2a within these two gradients at critical T i l growth cone decision points, and the frequency and type of erroneous axon projections that occur within the two gradients before and following perturbation of Sema 2a function, strongly establish that the shape of the Sema 2a gradient encodes the critical chemorepulsive guidance information to the pathfinding T i l growth cone. Furthermore, based on the exponential shape of the gradients and the sensitivity of the fractional change mechanism to the differences in gradient steepness, we propose that the T i l growth cone determines the direction and strength of the chemorepulsive force by detecting the fractional change in Sema 2a concentration across its spatial extent. To the best of our knowledge, this is the first functional analysis of the role of gradients in growth cone pathfinding and demonstration of a possible neuronal growth cone gradient-reading mechanism in vivo. 138 Discussion It is well established that gradients of molecules can guide neuronal growth cones in vitro; however, our understanding of these mechanisms has been limited by the scarcity of gradient data in vivo. We have used the development of the T i l pioneer neuron pathway within the grasshopper limb bud as a model system for investigating growth cone-gradient interactions in vivo. Perpendicular gradients specify target location. A criticism of the early versions of Sperry's chemoaffinity hypothesis has been that for every neuron to have a unique label, an unrealistic number of different proteins would be required to specify target locations. Sperry addressed these concerns by proposing that specificity could be attained by the superimposition of as few as two gradients. Indeed, this mechanism has been demonstrated in the retinotectal system where Eph receptors and their ligands are expressed in overlapping, complementary gradients within the retina and tectum (reviewed by Friedman and O'Leary, 1996; Braisted et al., 1997; Drescher et al., 1997). In the present study, we show that grasshopper T i l pioneer growth cones migrate down perpendicular gradients of the chemorepulsive secreted semaphorin Sema 2a, thus indicating that overlapping gradients of the same molecule can also achieve targeting specificity. The two repulsive Sema 2a gradients converge near the Tr guidepost cell located in the mid-trochanter. Interestingly, this is a position where the T i l pioneer growth cones stop and increase their sampling area, apparently searching for the appropriate cue(s) which will eventually reorient the growth cones to migrate ventrally within the trochanter. Blocking the function of Sema 2a leads to aberrant projections, where these aberrant growth cones often fail to contact the Tr guidepost cell, typically extend into the distal and dorsal limb compartments, and ultimately, fail to reach their targets within the CNS (Fig. 23; see also Isbister et al., 1999). We rarely observed aberrant projections into the ventral limb compartment, indicating the presence of an attractive cue emanating from the distal and dorsal limb bud region or, alternatively, a repulsive ventral cue. Regardless of additional cues, the perpendicular chemorepulsive gradients of Sema 2a are critical for the reliable extension of the T i l growth cones to the permissive mid-trochanter. The T i l pioneer growth cones migrate down these perpendicular chemorepulsive gradients towards an intermediate target, revealing that target specificity can be established by gradients in vivo. These findings are among the first to provide functional evidence for protein gradients in the establishment of accurate neuronal connectivity in the developing organism. Gradient shape confers guidance information to pathfinding growth cones. During two critical pathfinding decision points along the stereotyped T i l pioneer projection, the T i l growth cone interacts with gradients of Sema 2a that differ in steepness. Our analysis of the typical T i l growth cone pathfinding errors in normal developing grasshopper embryos revealed that the growth cones err more frequently during interaction with the shallow dorsal-ventral gradient of Sema 2a, than during interaction with the steep distal-proximal gradient. Our findings indicate that the higher degree of chemorepulsion exhibited by the distal-proximal Sema 2a gradient is conveyed by the steepness of the gradient, and not by the absolute level of protein. For a growth cone to use gradient shape for guidance, the growth cone must be capable of detecting spatial differences in guidance cues across its extent. A n elegant in vitro study by Baier and Bonhoeffer (1992) demonstrated that temporal retinal growth cones can detect small concentration changes of guidance molecules across their spatial extent. By varying the steepness of the repellent gradients in a stripe assay, they showed that the degree of growth cone response correlated with the strength, or steepness, of the gradient. In a subsequent study, Rosentreter et al. (1998) demonstrated that temporal retinal ganglion cell axons enter and extend up linear gradients of tectal membrane repellents to an avoidance point inversely correlated with 140 the slope; that is, axon extension is longer within shallower gradients. Notably, the points where axon extension stopped were not found at a common absolute concentration, but rather at a similar increment of concentration over the basal level. Although this study does not address the ability of gradients to induce growth cone turning, these findings are remarkably consistent with our in vivo results. We found T i l growth cones extend further up the shallow dorsal-ventral gradient of Sema 2a in comparison to the steep distal-proximal gradient. In addition, similar to the cessation of temporal retinal axon extension, the T i l growth cones likely turn to migrate down the repulsive gradients after detecting a critical increment, or fractional change, in Sema 2a across their spatial extent rather than an absolute change in concentration. Together, these studies demonstrate that neuronal growth cones can detect gradients of molecular cues and, in the present study, we provide in vivo evidence that the shape of the gradient confers the critical guidance information to the pathfinding growth cone. Growth cone gradient-reading mechanisms in vivo. For the shape of the Sema 2a gradient to confer guidance information, the gradient-reading mechanism employed by the T i l growth cone must be sensitive enough to detect the difference in steepness between the two gradients. We demonstrate that the fractional change mechanism is sensitive enough to detect the differences in steepness between the exponential distal-proximal and dorsal-ventral gradients, while the absolute change mechanism failed to detect the differences in gradient steepness. Research with leukocyte and bacterial chemotaxis indicates that these non-neuronal cells are sensitive to concentration changes as small as 1 % in the local environment (Devreotes and Zigmond, 1988). Our observations indicate that the differences in Sema 2a concentration detected across the T i l growth cone using the fractional change mechanism, however, are much larger ranging from 81 % in the distal limb bud to 51 % in the trochanter. Although the T i l growth cones may be capable of detecting smaller differences, 141 it is likely advantageous for the Sema 2a gradient to be expressed at levels considerably above the minimum detection level in order to ensure the accuracy of growth cone pathfinding. Interestingly, Rosentreter et al. (1998) demonstrated that temporal retinal axons in vitro adapted to differing basal concentrations of repellent, and subsequent extension up gradients was at a similar increment of concentration over the basal level. These results indicate that axon extension up repellent gradients is restricted by the amount of repellent encountered relative to a pre-adapted baseline. Furthermore, the temporal retinal growth cones did not use the concentration of repellent at or near their cell body as the baseline concentration, suggesting that the mechanism of adaptation likely resides within the growth cone. We propose that the T i l growth cone also exhibits a form of adaptation. When the T i l growth cones emerge from their cell bodies they are exposed to a high baseline of Sema 2a concentration and the fractional change in Sema 2a is calculated relative to this baseline. At the trochanter, however, the baseline of the dorsal-ventral gradient is considerably lower, and thus the T i l growth cone would have to adapt to this new baseline. The ability to adapt to input and reset its baseline may enable neuronal growth cones to continue pathfinding through regions of high repulsion. In the present study, we show that the chemorepellent Sema 2a is expressed in gradients within the developing grasshopper limb bud during the period of T i l pioneer neuron pathfinding. We took advantage of the large T i l growth cones and their well-characterized projection towards the CNS to investigate possible gradient-reading mechanisms in vivo. Our results indicate that the magnitude and shape of gradients differentially influence neuronal growth cones. The shape, or steepness, of the Sema 2a gradient confers the direction and strength of chemorepulsion, whereas the overall amount of Sema 2a encountered by the T i l growth cone appears to constrain its size. Furthermore, we provide evidence that the Ti 1 growth cone detects these gradients by measuring the fractional change in Sema 2a concentration across its spatial extent, thereby demonstrating one mechanism for growth cone gradient-reading in vivo. 142 V . G E N E R A L DISCUSSION The work presented in this thesis contributes to the expanding body of literature exploring mechanisms underlying the establishment of precise neuronal connectivity. We have used the T i l pioneer pathway within the developing grasshopper limb bud as a model system to investigate several aspects of growth cone guidance in vivo, including the role of candidate guidance cues and the mechanisms growth cones may employ to detect these cues. The role of adhesion in growth cone guidance. At the time of T i l pioneer neuron outgrowth, the grasshopper limb is a relatively simple structure consisting of epithelial cells, pre-axonogenesis neurons derived from the epithelium (termed guidepost cells) and mesoderm cells in the lumen of the limb. The grasshopper T i l pioneer growth cones migrate along the basal surface of the epithelium, on the epithelial side of the basal lamina, contacting the guidepost cells en route to the CNS. These substrates express a variety of substrate-bound guidance molecules, some of which have been shown to be necessary for accurate T i l growth cone pathfinding (Bentley and Caudy, 1983; Caudy and Bentley, 1986a; Kolodkin et. al., 1992; Sanchez et. al., 1995; Wong et. al., 1997). How these substrate-bound adhesion molecules interact with T i l filopodia to direct growth cone steering events in vivo is not well understood. Evidence from early in vitro experiments indicated filopodia may select the most adhesive pathway available for migration, supporting a model for growth cone steering based on differential expression of adhesive molecules in the environment (Letourneau, 1975). Similarly, studies in grasshopper suggested that substrate-bound adhesion molecules organized in a distal-proximal gradient of adhesiveness orient the T i l pioneer growth cones (Caudy and Bentley, 1986b; Condic and Bentley, 1989; reviewed by Bentley and O'Connor, 1992). An increase in filopodial:substrate adhesion of target-directed filopodia could lead to increased coupling of adhesion molecule receptors to the cytoskeleton and leading edge advance in these filopodia, thus steering the growth cone. We provide evidence that differential filopodial adhesivity does not predict T i l pioneer growth cone filopodial extension rate or steering events (Isbister and O'Connor, 1999). Our data confirms previous observations demonstrating an increase in filopodia: substrate adhesion at the trochanter segment epithelium. However, we establish that this increase in adhesion is uniform; consequently, the growth cone turning events and increases in filopodial extension observed at this decision point are not the result of differential filopodial adhesion across the growth cone. Therefore, while adhesive molecules are likely necessary for axon extension as well as for a variety of specific cell-cell interactions, differential adhesion is not the only force guiding neuronal growth cones towards their appropriate targets. At present, the majority of research is directed at elucidating the intracellular signalling components of cell adhesion molecules rather than considering their function simply in terms of adhesivity (Hynes, 1992; Wu et al., 1996; Burden-Gulley et al., 1997; Viollet and Doherty, 1997; Van Vactor, 1998). If the only role for substrate adhesivity were to ensure migrating growth cones remain closely adhered to the substrate, why would there be an increase in substrate adhesivity at the trochanter? Previous observations have shown that T i l growth cones radically alter their morphology upon migrating onto the trochanter epithelium (O'Connor et. al., 1990). Growth cones typically extend branches and filopodia along the trochanter epithelium, effectively increasing their sampling area along the dorsal-ventral axis (O'Connor et. al., 1990). It is possible that increases in substrate adhesivity at growth cone decision points, such as the trochanter segment epithelium, are responsible for these changes in growth cone morphology. A n alteration in growth cone morphology could effectively increase the probability of filopodial contact with guidance cues in the environment. For example, the secreted semaphorin gSema 2a is differentially expressed within the trochanter and detection of this molecule may be critical for 144 accurate T i l growth cone pathfinding. Therefore, one alternative role for substrate adhesivity may be to alter the morphology of growth cones so as to ensure filopodial interaction with guidance cues. The role of Semaphorins in Ti l pioneer axon guidance. In recent years many types of extracellular signals involved in growth cone pathfinding have been identified. They are presently categorized as either secreted or cell-associated molecules that can direct either attraction or repulsion (Tessier-Lavigne and Goodman, 1996; Goodman, 1996). Interestingly, some guidance molecules are bifunctional, eliciting attraction for some growth cones and repulsion to others (Colamarino and Tessier-Lavigne, 1995; Bagnard et al., 1998). Furthermore, this switching has been demonstrated within individual growth cones by altering levels of molecules such as cyclic nucleotides (reviewed by Caroni, 1998). One class of guidance molecules that may act as attractive and repulsive cues are the semaphorins, a large family of glycoproteins comprised of both secreted and cell-associated molecules (reviewed by Mark et al., 1997). In an effort to elucidate the role of semaphorins in neuronal growth cone guidance in vivo we have characterized a novel grasshopper secreted semaphorin, gSema 2a. Isolating grasshopper homologues of previously identified molecules has been particularly useful for studying the in vivo function of candidate guidance molecules at a cellular level. We demonstrate that Sema 2a acts as a chemorepulsive guidance molecule critical for determining both the initial direction and subsequent pathfinding events of the T i l axon projection. Our findings are consistent with the described roles for vertebrate secreted semaphorins, thereby providing another example of the conservation of developmental mechanisms across phyla. In addition to providing guidance information to the pathfinding T i l growth cone, several lines of evidence indicate that Sema 2a maintains axon fasciculation and constrains the growth 145 cone size: First, following Sema 2a perturbation we typically observed that the sibling axons were not fasciculated and that they often extended independent aberrant projections. Second, the T i l axons defasciculate more often within the trochanter, a region of shallow gradient expression of Sema 2a. In a similar fashion, it has been shown that temporal retinal axons grow more fasciculated on high concentrations of repellent membranes than on less repellent substrates. And third, with proximal migration towards the trochanter the T i l growth cone increases in size as the amount of Sema 2a expression decreases. The shallow gradient of Sema 2a within the trochanter may enable the T i l growth cones to defasciculate, branch and increase their sampling area, thus maximizing the likelihood of detecting the subsequent guidance cue(s) which induce the ventral turn. In fact, the dorsal-ventral gradient of Sema 2a may itself provide directional information and thereby contribute to the ventral turn. It is likely that Sema 2a provides a surround repulsion that encourages the T i l axons to fasciculate with each other, presumably a less inhibitory interaction. This preference to interact with neuronal substrates is also illustrated by the close apposition of T i l growth cones with the Tr and C x i cells, two preaxonogenesis neurons normally encountered along the T i l projection to the CNS (Caudy and Bentley, 1986). Furthermore, following Sema 2a perturbation, aberrant dorsal projecting growth cones were often observed to form close interactions with the neuronal Cx2 cells and femoral chordontal organ in the dorsal limb compartment, or even loop around to fasciculate with their own cell bodies. Our results suggest that aberrantly projecting T i l growth cones continue to prefer interactions with neuronal substrates rather than limb epithelium, indicating that even severely misguided growth cones continue to assess the relative attractiveness of their environment. The dynamic spatial and temporal expression of Sema 2a within the limb bud suggests multiple roles for this molecule during development. Following the completion of the T i l pioneer projection into the CNS, the expression of Sema 2a becomes restricted to more discrete 146 epithelial bands. These bands also appear to be graded, suggesting gradients may have a role in later grasshopper growth cone pathfinding events, or perhaps in refining target selection similar to the ephrins in retinotectal topography. Sema 2a is also uniformly expressed on certain muscles in the developing limb bud and may provide a more general inhibition for synapse promiscuity similar to the secreted Drosophila semaphorin, dSema 2a. Therefore, the pattern of Sema 2a expression likely provides distinct information to neuronal growth cones during different stages of axon outgrowth, target selection and synapse formation. During T i l pioneer axonogenesis and outgrowth (30 - 35% development), Sema 2a is expressed within the developing limb bud in overlapping distal-proximal and dorsal-ventral gradients which converge near the Tr guidepost cell within the trochanter limb segment. By approximately 32% development, transmembrane Sema l a is also detected in the trochanter and its expression pattern overlaps with the secreted semaphorin in the dorsal trochanter. Interestingly, it is at the mid-trochanter that the T i l growth cones abruptly halt their proximal extension, branch extensively, and sample both the dorsal and ventral trochanter before committing to a ventral turn. Consequently, both the secreted and transmembrane semaphorins are expressed during the period of T i l pioneer neuron pathfinding, and the expression pattern of these two semaphorins overlaps in a region where the T i l growth cone makes a critical steering decision. We found that the perturbation of both semaphorins leads to an increased frequency and severity of pathfinding errors, suggesting that the T i l growth cone receives simultaneous yet discrete information from the two semaphorins. These findings indicate that the T i l growth cone may express specific receptors for the two semaphorins, or that one receptor elicits distinct intracellular cascades in response to binding the different semaphorins. Neuropilins have been shown to function as components of a receptor complex for class III secreted semaphorins (Chen et al 1997; He and Tessier-Lavigne, 1997; Kolodkin et al., 1997); however, searches of the C. elegans and Drosophila genomes have not found any neuropilin 147 homologues. Recently, a novel member of the plexin family, PlexA, was isolated from Drosophila and demonstrated to bind transmembrane Drosophila semaphorins Sema 1 a and Sema lb (Winberg et al., 1998). Mutations in PlexA display the same motor and CNS phenotypes as Sema l a mutants and the two loci interact genetically, suggesting these genes are components of the same signalling pathway. These results are consistent with Plex A functioning as a neuronal receptor for the Drosophila transmembrane semaphorins. Plexin members are also found in vertebrates (Maestrini et al 1996; Kameyama et al, 1996; Satoda et al. 1995), however PlexA does not appear to bind the mammalian secreted semaphorins, nor has PlexA been demonstrated to bind the secreted Drosophila semaphorins. Although neither plexins nor neuropilins have yet been identified in grasshopper, the T i l pioneer pathway will likely provide a useful model for investigating possible receptor interactions of different semaphorin family members at the level of a single pathfinding growth cone. The balance of multiple signals guides neuronal pathfinding. During the period of T i l growth cone pathfinding, the gradient expression of Sema 2a in the ventral limb compartment ends at the trochanter, a region where Sema l a is expressed, and only the dorsal region of the limb bud continues to express high level of Sema 2a. The T i l growth cones typically halt their proximal migration within the Sema l a expressing trochanter epithelium and reorient ventrally, migrating away from the region of coexpression of Sema 1 a and Sema 2a in the dorsal trochanter. Our results demonstrate that the chemorepulsive gradient of Sema 2a effectively guides T i l growth cones to the Tr guidepost cell located within the band of Sema l a expressing epithelium. Following contact with the trochanter epithelia, however, Sema l a signalling appears to be critical for accurate T i l growth cone pathfinding. Given that contact of the T i l growth cones with the C x i neuron is also necessary for accurate pathfinding to 148 the CNS (Bentley and Caudy, 1983), then the T i l growth cones must respond to at least three distinct cues during development: Sema 2a, Sema la, and an unknown neuronal cell surface molecule. Thus, even in the relatively simple grasshopper model system, accurate axon outgrowth and pathfinding is dependent on the integration of multiple guidance cues by growth cones as they explore their environment. Intriguingly, perturbation of Sema 2a function rarely resulted in aberrant axon projection into the ventral compartment of the limb. One explanation for this is the possibility of an attractive cue in the dorsal and distal region of the limb, or a ventral repulsive cue, that Sema 2a is required to counter. Indeed, several molecules have been shown to be expressed in the insect limb during the period of pioneer axonogenesis and outgrowth (Bastiani et al., 1987; Norbeck et al., 1992; Diamond et al., 1993; Seaver et al, 1996). In the absence of Sema 2a repulsion, therefore, the T i l growth cones may project dorsally and/or distally in response to these cues. While the presence of additional attractive or repulsive signals is likely important for the pathfinding of other axons such as the distally projecting motor neurons, the T i l neurons must usually ignore these cues. Thus, under normal developmental conditions we suggest that it is the balance between multiple signals which maintains proximal T i l axonal extension along the limb axis towards the Tr cell. We found that the frequency of T i l projection defects was dependent on the amount of Sema 2a antibody included in the culture media. This dose-sensitivity provides further evidence for a fine balance among guidance signals and suggests that growth cones assess relative levels of attraction and repulsion during pathfinding. Our findings are similar to the observation that changes in gene dosage of dSema 2a and Drosophila Netrin B alter growth cone target selection by shifting the relative balance of inhibitory and attractive signals (Winberg et al., 1998). Thus, Drosophila Sema 2a does not prevent growth cones from exploring their environment, but establishes a threshold that specific attractive signals must overcome in order to permit synapse formation. Taken together, our axon initiation and pathfinding results and the 149 genetic analysis of guidance molecule interactions during motor neuron target selection and synapse formation, illustrate that all stages of axonal development are dependent on the reception and comparison of multiple guidance cues. Axon guidance by gradients of chemorepulsion. In general, growth cones are guided to their targets by at least two broad mechanisms: contact-mediated and chemotactic (reviewed by Nieto, 1996; Goodman, 1996; Varela-Echavarria and Guthrie, 1997). Cell-associated molecules are proposed to guide growth cones via short-range, contact-mediated mechanisms, such as adhesion and its resulting downstream intracellular signalling cascades. In contrast, the diffusible chemoattractive and chemorepulsive guidance molecules have been proposed to exert their biological effect over longer distances by establishing gradients of protein distribution. However, only recently has the existence of protein gradients been directly demonstrated in vivo (Norbeck et al., 1992; Monschau et al., 1997; Isbister et al., 1999). The scarcity of in vivo gradient data has limited our understanding of gradients and the mechanisms growth cones employ to read them. Our demonstration that gradients of the chemorepulsive guidance cue Sema 2a are critical for directing T i l pioneer growth cones towards the CNS provides important functional evidence for the role of gradients in neuronal growth cone guidance in vivo. At present, the regulation of Sema 2a protein expression is unknown. gSema 2a and its message are colocalized, suggesting that although the protein is secreted it does not diffuse far and, therefore, the resulting protein gradient is likely established at the transcriptional level. This immobilization of a diffusible guidance molecule close to its point of secretion illustrates that short-range and long-range guidance mechanisms can be indistinguishable. Secreted guidance molecules can, therefore, range from being freely diffusible to cell-associated, and thus create 150 varying expression patterns from gradients to sharp boundaries. We use our in vivo gradient data to explore whether information can be encoded by variable guidance molecule expression pattern. We show that the T i l growth cones will extend into both the steep and shallow gradients of Sema 2a, however, the steering events are more reliable when the growth cone is interacting with the steep gradient. Therefore, discrete growth cone guidance information can indeed be encoded in the pattern of protein expression, as steeper gradients of Sema 2a confer more chemorepulsion to pathfinding T i l growth cones. Why guide growth cones with gradients of repulsion? One possibility is that gradients allow degrees of inhibition. By not completely collapsing the growth cone upon contact, gradients permit the continued sampling of environment and continued migration. For example, though repelled by areas of high expression, the T i l growth cones are still able to grow on a substrate of Sema 2a. Similarly, cultured temporal retinal axons are inhibited from branching on an incorrect target consisting of posterior tectal membranes, yet they are still able to extend across these membranes (Roskies and O'Leary, 1994). In fact, the strict avoidance of repellent membranes by temporal retinal axons in vitro was observed only at the border of very high steps of repellent tectal membrane (Baier and Bonhoeffer, 1992). We speculate that the high levels of Sema 2a in the extreme distal tip may be sufficient to collapse the T i l growth cone, creating an exclusion zone. This complete collapse would be reasonable as there is no apparent benefit to sampling this region. However, i f this high Sema 2a expression started immediately surrounding the cell bodies, the growth cones could be prevented from emerging altogether. Therefore, to ensure both reliable T i l axon initiation and proximal extension, the optimal shape for the Sema 2a gradient in the distal limb bud would be a steep gradient decreasing from high levels in the distal tip to a plateau surrounding the T i l cell bodies. Conversely, a more shallow dorsal-ventral chemorepulsive gradient of Sema 2a within the trochanter would provide the necessary chemorepulsion to minimize dorsal projection errors, yet 151 still be shallow enough to permit growth cone expansion and exploration of both the dorsal and ventral limb compartments. Thus, by encoding information in the shape of the gradient even overlapping gradients of the same molecule can present different levels of repulsion. Growth cone gradient-reading mechanisms. For a growth cone to be guided by a gradient, it must be able to sense a sufficiently large difference in ligand concentration over its spatial extent. Two theoretical models for the deciphering of gradient information by growth cones have been proposed and they differ on which aspect of the change in ligand concentration is most critical (Goodhill and Baier, 1998; Goodhill, 1998). The absolute change model determines the absolute change across the growth cone, whereas the fractional change model calculates the change as a fraction of the maximum concentration detected at one edge (for more detailed description of these models, see Results and Discussion of Chapter 4). Similar models are under debate in other eukaryotic cell types (reviewed by Devreotes and Zigmond, 1988; Parent and Devreotes, 1999); however, the lack of gradient data in vivo has limited our investigation of these possibilities for neuronal growth cones. We provide evidence that the T i l growth cone employs the fractional change mechanism to read the chemorepulsive gradients of Sema 2a. To the best of our knowledge, this is the first demonstration of a possible neuronal growth cone gradient-reading mechanism in vivo. In vitro, it has been demonstrated that temporal retinal axons enter and extend up linear gradients of tectal membrane repellents to an avoidance point which is inversely correlated with the slope, that is, axon extension is longer within shallower gradients (Rosentreter et al., 1998). The points where axon extension stopped were not found at a common absolute concentration, but rather at a similar increment of concentration over a pre-adapted basal level. These in vitro axon extension findings are consistent with our in vivo growth cone steering results. We found T i l growth cones extend further up the shallow dorsal-ventral gradient of Sema 2a in comparison 152 to the steep distal-proximal gradient. In addition, similar to the cessation of temporal retinal axon extension, the T i l growth cones likely turn to migrate down the repulsive gradients after detecting a critical fractional change in Sema 2a across their spatial extent rather than an absolute change in concentration. Regardless of the mechanism a growth cone employs to read a chemotactic gradient, the gradient must satisfy at least two physical constraints to provide reliably guidance to a pathfinding growth cone. First, the absolute concentration of guidance cue must not be too high or too low. If the concentration of the guidance cue is too high, nearly all the receptors will be bound most of the time, resulting in little difference in binding across the extent of the growth cone. Conversely, i f the concentration is too low, almost none of the receptors will be bound, again resulting in little difference across the growth cone. Given that the T i l growth cone obtains guidance information from both the shallow and steep gradients of Sema 2a by detecting a difference in Sema 2a across its spatial extent, we predict the absolute expression levels of Sema 2a observed in the limb are appropriate for the T i l growth cone Sema 2a receptor pool. The second physical limitation on the distribution of a chemotactic molecule is that for a growth cone to reliably detect the difference in concentration across its spatial extent, the change in concentration must be large enough to overcome the noise inherent to receptor binding and intracellular signalling. By establishing gradients of a repellent and measuring the response of retinal axons, Baier and Bonhoeffer (1992) estimated the minimum fractional change detectable by a growth cone to be 1%. This is consistent with studies of cell chemotaxis in various systems which have suggested optimal values of 2% (Devreotes and Zigmond, 1988). Using our in vivo gradient data we calculated the difference in Sema 2a concentration across the Ti 1 growth cone to range from -50% - 80%, values considerably higher than the predicted minimum detection level. Although the T i l growth cone may be sensitive to concentration changes as small as 1 -2%, growth cone pathfinding may be more reliable when the change in Sema 2a concentration is above the minimum detection level. 153 Therefore, both the absolute concentration of the cue and the steepness of the gradient are important limitations on the chemotactic guidance of growth cones. We demonstrate that Sema 2a expression satisfies these constraints, exhibited by the reliable T i l pathfinding in response to these gradients; however, the critical guidance information is encoded by the steepness of the gradient, rather than the absolute concentration. How are chemotactic gradients translated into directional growth cone migration? Filopodia direct growth cone steering by integrating and transducing the guidance information from multiple external cues into motile forces (reviewed by Heidemann et al., 1990; Lin et al., 1994; Kater and Rehder, 1995; Suter and Forscher, 1998). Ultimately, for directed neurite outgrowth to occur, the guidance information detected by filopodia must be converted into an intracellular signal that regulates the growth cone cytoskeleton. Previous observations in grasshopper have suggested that distal-proximal gradients of adhesive molecules were responsible for the proximal extension of T i l pioneer axons towards the CNS (Caudy and Bentley, 1986a; Condic and Bentley, 1989b; Condic and Bentley, 1989b). However, we have established that adhesion alone is insufficient to guide T i l pioneer growth cone steering events (Isbister and O'Connor, 1999) and, therefore, molecular gradients likely guide growth cone pathfinding through alternative mechanisms. Weiner et al. (1999) have recently shown that external chemotactic gradients cause neutrophils to organize new sites of actin polymerization on the cell surface directed towards the highest concentration of chemoattractant. Furthermore, these sites of actin polymerization co-localize with Arp2/3, a complex known to stimulate actin polymerization and to directly link signal transduction pathways to the actin cytoskeleton (Machesky and Gould, 1999; Rohatgi et a l , 1999). Thus, it is proposed that chemotactic 154 gradients produce directional migration through differential activation of intracellular signalling cascades and the spatial regulation of actin dynamics. A similar spatial regulation of actin polymerization may underlie T i l growth cone steering events during interaction with the chemorepulsive gradients of Sema 2a. We have observed that T i l pioneer growth cones in the process of turning ventrally within the trochanter extend ventrally-directed filopodia 3-times faster than dorsally-directed filopodia (Isbister and O'Connor, 1999). Surprisingly, this differential extension rate is evident even when ventrally-directed filopodia are compared to dorsally-directed filopodia of the sibling T i l growth cone, even though these filopodial populations overlap and sample the same environment only in reverse orientation. We speculate that the increased extension rate of ventrally-directed filopodia results from the T i l growth cone detecting the direction of the dorsal-ventral Sema 2a gradient and translating this gradient information into preferential localization of actin polymerization sites to those filopodia directed down the chemorepellent gradient. The T i l pioneer projection and chemorepulsive gradients of Sema 2a will likely provide a useful model system for further analysis of growth cone gradient-reading mechanisms and intracellular responses to chemotaxis in vivo. Summary of possible mechanisms guiding the Ti l pioneer projection. Based in part on the information presented in this thesis, we have formed the following working hypothesis of the some of the possible molecules and mechanisms involved in T i l pioneer growth cone pathfinding to CNS. (1) The steep distal-proximal chemorepulsive gradient of Sema 2a ensures proximal T i l pioneer axonogenesis. (2) Following axonogenesis, the overlapping chemorepulsive distal-proximal and dorsal-ventral gradients of Sema 2a maintain axon fasciculation and effectively guide the T i l growth cones to the Tr guidepost cell located 155 within the trochanter epithelium. (3) Within the trochanter, the increased adhesivity, possibly conferred by the transmembrane Sema la, combined with less inhibition from Sema 2a, leads to cessation of the proximal extension of the T i l growth cones, increased growth cone branching and increased filopodial exploration. (4) The predominant dorsal-ventral gradient of Sema 2a within the trochanter may contribute to the ventral turn of the T i l growth cones. (5) Contact with an as yet unidentified positive cue on the C x i guidepost cells enables the T i l growth cones to exit the adhesive trochanter, followed by continued proximal migration into the CNS. We further propose that the steepness of the Sema 2a gradients, detected as a fractional change across the growth cone, confers the critical guidance information; whereas the absolute concentration constrains the morphology of the growth cone. Therefore, along the T i l pioneer pathway semaphorins provide both contact-mediated and chemorepulsive guidance information and act in combination with a variety of other molecules to effectively guide T i l pioneer growth cones into the CNS. Conclusion For directed axon outgrowth to occur, multiple signals must be continuously combined and integrated within the pathfinding growth cone. 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