Department of Cell Biology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06510, USA
*Author for correspondence (e-mail: mtiemeyer{at}glyko.com)
Accepted August 16, 2001
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SUMMARY |
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Key words: Carbohydrate, Lectin, Axon pathfinding, Glia, Drosophila
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INTRODUCTION |
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The function of a population of glial cells at the Drosophila embryonic midline (midline glial cells) is crucial to the guidance and organization of commissural and longitudinal fibers (Hummel et al., 2000; Hummel et al., 1999). Signals originating from the midline glia allow commissural axons to cross the midline and keep longitudinal axons away from the midline (Harris et al., 1996; Seeger et al., 1993; Stein and Tessier-Lavigne, 2001). Both the amount (dose) and the type (molecular identity) of signaling can determine growth cone behavior, dictating the co-requisite establishment of an axon-glia interface that facilitates high-fidelity transmission of molecular signals (Kidd et al., 1998b; Winberg et al., 1998). While the identities and functions of relevant signaling molecules and receptor families have been elegantly described, comparatively little progress has been made towards understanding whether additional components of the neuronal and midline glial cell surfaces contribute to signal transmission by regulating the axon-glia interface.
At the very least, it may be necessary for exploring growth cones to adhere to the midline glial surface before sufficient signal integration or sorting ensures that the maturing axon is appropriately routed. Among Drosophila molecules that mediate cell adhesion, only the Gliolectin protein is expressed in midline glia, coincident with the extension of commissural and longitudinal axon pathways (Tiemeyer and Goodman, 1996). Originally identified in an adhesion-based cloning screen for embryonically expressed carbohydrate-binding proteins (lectins), Gliolectin binds a subset of N-acetylglucosamine-terminated Drosophila glycans. That a lectin-carbohydrate interaction might contribute to the initial contact between axons and midline glia is consistent with demonstrated and proposed functions of carbohydrate-mediated cell adhesion in other contexts.
The tissue, cell-type and developmental specificity of glycan expression has engendered proposals that cell-surface oligosaccharides function as recognition ligands. The characterization of Drosophila glycans is still in its infancy but so far indicates complexity sufficient to support cell-specific expression comparable to that of other organisms (Callaerts et al., 1995; DAmico and Jacobs, 1995; Fredieu and Mahowald, 1994; Fristrom and Fristrom, 1982; Rietveld et al., 1999; Roth et al., 1992; Seppo et al., 2000; Seppo and Tiemeyer, 2000; Toyoda et al., 2000). We have therefore investigated whether loss of Gliolectin generates phenotypes indicative of aberrant cell-recognition at the midline. We find that loss of Gliolectin affects the efficiency but not the specificity of pathfinding. While gross alterations in the architecture of the axon scaffold are apparent, commissures and longitudinals still form in the absence of Gliolectin. The results are consistent with a need for carbohydrate-mediated axon capture at the midline that initiates and facilitates subsequent signaling between axon and glia. We propose that specific axon pathfinding across the midline emerges from combining axon capture, which may be largely indiscriminate, with subsequent signal transmission.
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MATERIALS AND METHODS |
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Fly stocks
The rhomboid-Gal4 line, w; rho-Gal4/rho-Gal4, was obtained from Tian Xu, Yale University. The Gliolectin null line Df(3R)eBS2, rsd1/TM3 (the deletion chromosome is designated 3013) and all other stocks were acquired from the Bloomington Stock Center (Bloomington, IN). For generation of UAS-gliolectin (UAS-glec) transformant lines, a XhoI fragment, containing the entire Gliolectin-coding sequence, was cut from subclone NH3-2/19 in pCDM8 and ligated into pBluescript (Tiemeyer and Goodman, 1996). Subcloned plasmid bearing Gliolectin insert in the appropriate orientation was then digested with NotI and KpnI, and the resulting Gliolectin fragment was ligated into the pUAST3 vector previously cut with the corresponding enzymes (Brand et al., 1994). Injection of the resulting construct, designated pUAST3glec, into w1118 flies and the generation of transformant lines were achieved using standard procedures.
Immunohistochemistry
Embryo collections were dechorionated, fixed and devitellinized according to standard methods (Patel, 1994). Because decreased 1B7 (anti-Gliolectin) staining was observed in embryos stored for extended periods (> 1 month) in methanol at 20°C, embryos were routinely processed for staining within 2 weeks of preparation. After two 10 minute washes in PBT (0.3% w/v Triton X-100 in phosphate-buffered saline (PBS)), embryos were blocked for 30 minutes in PBT containing 0.1% w/v bovine serum albumin and 5% normal goat serum at room temperature. Dilutions of primary antibody in the same solution were then substituted for blocking solution and the embryos were incubated at 4°C overnight. Primary antibody was washed out with four 10 minute washes in PBT at room temperature. After reblocking for 15 minutes, embryos were incubated at room temperature for 3 hours in horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody diluted 1:500. After washing out the secondary antibody, HRP activity was detected by precipitation of DAB (3-3' diaminobenzidine, 0.3 mg/ml in PBT) in the presence of H2O2 (0.03% w/v). After 15 minutes in the detection reagent, embryos were washed and subsequently cleared by settling through 50%, then 70% glycerol in PBS. In those cases where the DAB reaction product was nickel enhanced, NiCl2 (0.64% w/v final concentration) was added prior to the addition of H2O2. Primary antibody dilutions were 1:5 for 1B7, 1:3 for BP102 and 1:50 for 1D4. After staining, embryos were staged as whole mounts and then dissected using tungsten needles before photomicroscopy.
For frozen sections, stained embryos were equilibrated overnight in SPT (25% w/v sucrose, 0.03% w/v Triton X-100 in PBS) immediately after the detection reaction. After staging as whole mounts, the desired embryos were segregated and pipetted in 20 µl total volume of SPT into the top 25% of a small volume (approximately 200 µl) of TBS Tissue Freezing Medium (Electron Microscopy Sciences) in a 1.5 ml eppendorf centrifuge tube. As the embryos sank through the mounting media, they oriented themselves vertically along their anterior-posterior axis. Once the embryos were sufficiently aligned, the entire centrifuge tube was quick frozen in crushed dry ice. The conical part of the centrifuge tube was then clipped just above the position of the embryos, releasing a small, frozen cone of mounting media (approximately 100 µl) containing the aligned embryos. After attachment to a cryostat chuck, serial 20 µm sections through multiple segments of multiple wild-type and 3013/
3013 embryos were collected on clean glass slides. After drying overnight at room temperature, the sections were coverslipped under 50% glycerol before photomicroscopy.
Embryo staging is as described by Campos-Ortega (Campos-Ortega and Hartenstein, 1997) as modified by Klämbt (Klämbt and Goodman, 1991b). Stages 12/5 through 12/0 were identified by the number of segments remaining on the dorsal side of the retracting germ band. Stage 13 was judged to be early when the amnioserosa was completely unfolded, the clypeolabrum was extremely thin and the hindgut had not yet assumed a sigmoidal shape. The stages from mid-13 to early 14 were distinguished by the formation of the dorsal ridge, by the extent of the dorsal expansion of the lateral ectoderm, and by the morphology of the hindgut and of the maxillary, mandibular and labial segments.
In rescue experiments, multiple landmarks were used to distinguish transgenic embryos lacking endogenous Gliolectin (UAS-glec/+; rho-Gal4/+; 3013/
3013) from those that carried a functional copy of the glec gene (supplied by the balancer chromosome). By mid-stage 13, Rho drives expression in mesodermal and ectodermal cells that do not normally express Gliolectin protein (Bier et al., 1990). In addition to the midline glia of the ventral nerve cord, Glec protein is also normally expressed by a subset of cells in the supraesophageal ganglia. Therefore, absence of 1B7 staining in the head, accompanied by staining of all midline glia and additional ectodermal and mesectodermal cells indicates transgenic Gliolectin expression in a Glec-null embryo. When embryos were double stained for both Gliolectin expression (1B7) and an axonal marker (1D4 or BP102), it was necessary to nickel enhance the axonal marker (black) so that it could be clearly distinguished above the 1B7 midline staining.
Generation of rescue lines
Transformation with the pUAST3glec vector yielded UAS-glec insertions on both the X (line 8-19) and second chromosomes (line 10-9). To introduce the UAS-glec element into the gliolectin null background, the 3013 deletion line was first crossed to w; D,ry/TM3 to put the deficiency into a w background. The resulting w;
3013/TM3 stock was crossed to the UAS-glec insertion line 8-19 to create a UAS-glec;
3013/TM3 stock. Similarly, w; rho-Gal4/ rho-Gal4 (rhomboid-Gal4) flies were crossed to
3013 to create w; rho-Gal4/+;
3013/TM3 stocks. It was not possible to generate flies homozygous for rho-Gal4 in this background. Single-copy rescue was assessed by collecting embryos from a cross of UAS-glec;
3013/TM3 females to w; rho-Gal4/+;
3013/TM3 males. For double-copy rescue, a UAS-glec; UAS-glec;
3013/TM3 stock was generated from UAS-glec insertion lines bearing elements on the X (line 8-19) and second chromosomes (line 10-9).
Generation of EMS mutants
Male w1118 flies (n=50) were treated with ethylmethanesulfonate (EMS) according to standard methods (Lewis and Bacher, 1988). Mutagen-treated males were mated to virgin w; D,ry/TM3 females. Single male or female w, Sb, D+ progeny were then mated to either w; 3013/TM3 or w;
3013/TM6b (n=600). Ten of these crosses gave only Sb (TM3-balanced) or Hu (TM6b-balanced) progeny, indicating non-complementation of the
3013 lethality, and were kept for further analysis.
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RESULTS |
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Pioneering of longitudinal pathways is delayed by loss of Gliolectin
The formation of longitudinal pathways is pioneered, in part, by the extension of the pCC and vMP2 axons from each segment towards the next anterior segment (Jacobs and Goodman, 1989a; Jacobs and Goodman, 1989b). Longitudinal extension of these axons is guided by growth cone repulsion from the midline, by positive interactions with laterally positioned intermediate targets (SP1 neurons and longitudinal glia) and by specific fasciculation with posteriorly-extending longitudinal processes (MP1 and dMP2). Several of the cells involved in longitudinal pathfinding are visualized with mAb 1D4 (anti-Fasciclin II), including pCC, vMP2, dMP2, MP1 and SP1 (Goodman and Doe, 1993). Early in longitudinal pathfinding (stage 12/1), the pCC axon does not extend as far anteriorly in 3013 homozygotes or in glecm98/
3013 embryos as in wild type (Fig. 5A,D); rather, it appears stalled after minimal outgrowth (Fig. 5G,J). Later (stage early 13), while the pCC/vMP2 process in wild type has completed its extension to the next segment (Fig. 5B,E), it frequently remains stalled and is often in intimate contact with 1D4-positive midline cells (MP1 and dMP2) in deletion embryos (Fig. 5H). In glecm98/
3013 embryos, formation of the pCC/MP1 pathway is severely hampered (Fig. 5K) with few pCC cells showing extension beyond the segment of origin. In slightly older
3013 homozygous embryos (stage late 13), many segments exhibit near normal (Fig. 5C,F) extension of pCC to the next anterior segment (Fig. 5I). However, glecm98/
3013 embryos continue to lack a formed pCC/MP1 pathway at this stage (Fig. 5L). Almost all
3013/
3013 embryos (95%) exhibited delayed longitudinal growth in, on average, more than one-half of their segments (Table 1). Longitudinal pathfinding defects were not seen in glecm24/
3013 embryos. The segmental broadening observed with mAb BP102 (Fig. 2, Fig. 3) was not apparent with mAb 1D4.
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Pathfinding defects are rescued by restoration of Gliolectin expression at the midline
To establish that fused commissure and delayed longitudinal phenotypes are attributable to altered glec function, Gliolectin was transgenically expressed (UAS-glec) at the midline of 3013/
3013 embryos under indirect control of the rhomboid promoter (rho-Gal4). As rho is normally activated in cells that also express Gliolectin, rho-induced Glec closely reconstitutes wild-type expression (Bier et al., 1990). Although introduction of one copy of the UAS-glec transgene did not rescue the embryonic lethality associated with
3013 homozygosity, it did result in significant improvement in both the commissural organization and longitudinal outgrowth phenotypes (Table 1). Commissural separation is more distinct (Fig. 6A-C) and the intersegmental extension of longitudinal pioneers approaches completion on schedule (Fig. 6E-G, arrow). However, neither phenotype is completely rescued with only one copy of the UAS-glec transgene in
3013/
3013 embryos. Decreased longitudinal mass (Fig. 6C, arrow) appears more resistant to rescue than commissural fusion (Fig. 6C, arrowhead). A single copy of UAS-glec completely rescues the axonal phenotypes of both glecm24/
3013 and glecm98/
3013 (Table 2) and transgenic Gliolectin expression rescues the lethality of both EMS mutants in the
3013 background. Construction of
3013/
3013 embryos in which a single rho-Gal4 element drives expression of Gliolectin from two copies of UAS-glec yields nearly complete rescue of delayed longitudinal outgrowth (Fig. 6H, arrow), decreased longitudinal mass (Fig. 6D, arrow) and commissural disorganization (Fig. 6D, arrowhead) associated with the loss-of-function phenotype (Table 1).
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DISCUSSION |
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Loss of Gliolectin affects the efficiency of midline signal transmission
Axons that pioneer longitudinal pathways, such as the axon of the pCC neuron, are normally retained on the ipsilateral side of the nerve cord by repulsive signals expressed by the midline glia. Interactions between Slit, a midline glial protein, and neuronal Roundabout (Robo) receptors maintain longitudinal axon growth in an anterior-posterior trajectory by causing the withdrawal of nascent contacts between longitudinal axons and the midline. Faced with a formidable inhibitory environment at the midline, susceptible axons efficiently extend along more favorable longitudinal routes. In the absence of repulsive signaling components (either Slit or Robo), axons normally destined to remain in the longitudinals instead freely cross the midline, indicating that the midline possesses an underlying substrate favorable for axon growth (Kidd et al., 1999; Kidd et al., 1998a; Rajagopalan et al., 2000).
In the absence of Gliolectin, we find that midline repulsion of longitudinal axons is not abolished. However, as demonstrated by the behavior of the pCC axon, the efficiency of the repulsive response is reduced. We observe that the pCC axon is delayed in its anterior course, frequently exhibiting extended interactions with what are usually transient substrates (Fig. 7). Such aberrant behavior indicates that Gliolectin normally provides sufficient contact between midline glia and filipodial extensions to ensure efficient repulsive signal transmission. Therefore, the observed glec phenotype predicts that partially penetrant midline-crossing defects associated with robo hypomorphic alleles should be enhanced by a concomitant reduction in Gliolectin.
Axons that pioneer commissural pathways normally extend through the midline by ignoring repulsive signals (Seeger et al., 1993). The activity of the Commissureless (Comm) protein, another midline glial protein, instructs a subset of axons to downregulate Robo receptors and consequently disregard Slit (Tear et al., 1996). Loss of the Commissureless protein results in loss of commissures because axons are uniformly repelled from the midline. However, despite loss of this inhibitor of repulsion, axons initially orient towards and probably also adhere to the midline glia (as these cells are laterally displaced in some comm mutant alleles). Adhesion, or at least intimate contact, between presumptive commissural axons and the midline glia is also implicit in the proposal that Comm signaling proceeds through the transcellular transfer of Commissureless transmembrane protein from midline glial cell to axon (Tear et al., 1996).
As loss of Gliolectin affects the extent of physical contact between axons and midline glial cells (Fig. 7), minimal Commissureless signaling would be expected in glec mutants. Therefore, loss of Gliolectin should enhance hypomorphic comm alleles by attenuating residual signaling. Furthermore, as axon commissures are present, although distorted, in glec embryos, it is once again apparent that loss of Gliolectin affects the fidelity of axon fasciculation and not the absolute specificity of pathfinding. In the absence of the adhesivity provided by Gliolectin-carbohydrate interactions, the amount of signaling passed between the midline glia and a neuronal growth cone is controlled solely by the duration of chance filipodial contact. Gliolectin functions, therefore, as a capture mechanism that ensures neural and glial membranes remain in contact long enough to communicate and integrate relevant sorting information. Recently, an adult bristle phenotype was described in an EP line possessing an Enhancer/Promoter P-element insertion into the glec locus, suggesting that in other contexts Gliolectin may also facilitate cell signaling (Abelilah-Seyfried et al., 2000).
Gliolectin function is consistent with other carbohydrate-mediated interactions
In several contexts, carbohydrate-binding proteins and their recognized ligands provide a mechanism for capture at specific tissue sites. For example, the Selectin family of C-type lectins and their carbohydrate ligands are expressed on leukocytes, platelets and endothelial cells where they mediate the initial interactions between inflammatory cells and specialized endothelial domains in vertebrate vasculature (Tiemeyer et al., 1991; Whelan, 1996). Circulating lymphocytes are recruited to sites of endothelial inflammation by the interaction between a lymphocyte-expressed selectin (L-Selectin) and appropriate endothelial cell carbohydrate ligands (primarily oligosaccharides bearing a sialyl-LewisX structure). Engagement of sufficient selectin-ligand pairs induces lymphocytes to roll along the endothelium, thus allowing additional signaling and adhesive interactions to generate subsequent cellular extravasation (Springer, 1994). At the outset of this process, it is the presence of a carbohydrate-mediated capture mechanism that ensures the efficiency and localization of the inflammatory response.
As loss of selectin-mediated interactions abolishes leukocyte extravasation while loss of Gliolectin does not completely abolish normal axon sorting, the parallels between selectin-mediated whole cell capture and Gliolectin-mediated axon capture are not absolute (Maly et al., 1996). Selectins, however, operate under conditions of hydrodynamic flow where cells are quickly swept away if not captured. The environment in which Gliolectin operates is more static; extending axons reside in appropriate regions of the developing nerve cord long enough to heed available signals, despite the loss of intimate glial contact. Under these conditions, loss of Gliolectin affects only the efficiency not the direction of axon pathfinding. Nonetheless, the important characteristic shared by Gliolectin- and Selectin-mediated carbohydrate binding is that they constitute the initial step in a process that leads to subsequent cellular responses (Lasky, 1992; Lasky, 1995).
For longitudinal axons, this response is withdrawal from the midline. For commissural axons, the response to Gliolectin-mediated contact is to maximize contact with midline glial cells at the expense of axon-axon contact. Gliolectin, then, provides a permissive substrate for commissural axon segregation. Similarly, in the vertebrate embryonic nervous system, regulated expression of an anionic carbohydrate polymer, poly-2,8-linked sialic acid, facilitates segregation of motor axons into functionally related fascicles (Acheson et al., 1991; Stoeckli et al., 1997). Furthermore, the fidelity of axon fasciculation in developing invertebrate nervous systems is affected both by altered glycan expression and by glycan-directed biochemical perturbations (Song and Zipser, 1995; Whitlock, 1993). While these glycan-mediated axon segregation events have not been shown to require a carbohydrate-lectin interaction, they nevertheless demonstrate the importance of regulated carbohydrate expression and oligosaccharide function in axon sorting.
Carbohydrate-mediated axon capture functions within a hierarchy of activities to ensure appropriate axon pathfinding
It was first suggested over 30 years ago that carbohydrate-protein interactions might impart specificity to cell-cell interactions (Roseman, 1970). The nervous system, which is particularly rich and varied in its glycan expression, has historically served as a hunting ground for signs of carbohydrate-mediated cell-cell recognition, especially during development. Characterization of the diversity and intricacy of neural cell-specific glycan expression continues to expand. However, a corresponding range of endogenous neural lectins with the capacity to interpret the cell-surface carbohydrate code has yet to be described. Although perhaps simply reflecting the relative paucity of genetic and molecular analysis applied to carbohydrate function, this deficiency also reinforces the suggestion that specificity is primarily an emergent property of the interdependent activities of multiple genes. Even though Gliolectin function is necessary to efficiently initiate a cascade that results in specific pathfinding, axon capture by itself does not guarantee appropriate growth cone response. Likewise, normal signaling in the absence of axon capture yields imperfect fasciculation.
Thus, on top of regulated signaling molecule and receptor function, the embryonic nervous system overlays appropriate spatial and temporal expression patterns of lectin-ligand pairs to ensure high-fidelity axon pathfinding. Estimating the total number of lectin-ligand pairs relevant to Drosophila embryonic development would be excessively speculative even with completion of the genome sequence (Dodd and Drickamer, 2001; Drickamer and Dodd, 1999; Seppo and Tiemeyer, 2000; Theopold et al., 1999). However, by wedding signaling specificity to carbohydrate-mediated capture, the need for stringent recognition markers is reduced. Additionally, if non-interacting cells effectively ignore locally proffered signals, a small number of moderately discriminate adhesion molecules would reduce the level of specificity required of signaling mechanisms. The composite effect, then, of carbohydrate-mediated axon capture would be to sharpen the sphere of influence of combinatorial signaling codes and thus impart greater specificity to axon pathfinding.
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ACKNOWLEDGMENTS |
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