Heterodimeric cell surface receptor integrin is widely expressed in the nervous system, but its specific role during axon development has not been directly tested in vivo. We show that theDrosophila nervous system expresses low levels of positron-specific (PS) integrin subunits αPS1, αPS2, and βPS during embryonic axogenesis. Furthermore, certain subsets of neurons express higher levels of integrin mRNAs than do the rest. Null mutations in either the αPS1 or αPS2 subunit gene cause widespread axon pathfinding errors that can be rescued by supplying the wild-type integrin subunit to the mutant nervous system. In contrast, misexpressing either the αPS1 or αPS2 integrin subunit in all neurons leads to no obvious axon pathfinding errors. We propose that integrin does not itself serve as either a “clutch” constituting molecule or a specific growth cone “receptor,” as proposed previously, but rather as part of a molecular network that cooperatively guarantees accurate axon guidance.
- axon guidance
- growth cone
- multiple edematous wings
Axon guidance relies on the activation of cell surface receptors that translate extrinsic cues into directed cytoskeletal rearrangement within a growth cone (Chiba and Keshishian, 1996; Goodman, 1996; Jay, 1996). Recent studies establish that many members of integrin, a cell adhesion/signaling molecule, are widely distributed in developing nervous systems (Schmidt et al., 1995;Varnum-Finney et al., 1995; Jones, 1996; Lallier et al., 1996; Martin et al., 1996; Shaw et al., 1996; Wu et al., 1996; Grotewiel et al., 1998). In vitro studies show that integrin is enriched at the tip of a growing axon, suggesting a role in axon guidance (Wu et al., 1996; Grabham and Goldberg, 1997; Takagi et al., 1998). One model proposes that integrin is an important component of the “clutch,” a molecular complex that is responsible for growth cone advancement by anchoring cytoplasmic actin filaments to the extracellular substrate (C. H. Lin et al., 1994; Schmidt et al., 1995). Another model predicts that integrin serves as a cell-specific recognition “receptor” that biases growth cone movement toward the ligand source (Kuhn et al., 1995, 1998). These ideas have not been testedin vivo.
We chose Drosophila as a model system for studying integrins because its nervous system consists of well-defined axon pathways (Chiba, 1998) and because it has a small set of integrin subunits similar in structure and function to those in vertebrates (MacKrell et al., 1988; Leptin et al., 1989; Wilcox, 1990; Brown, 1993, 1994; Yee and Hynes, 1993; Gotwals et al., 1994a,b; Brower et al., 1995; Bunch et al., 1998). A positron-specific (PS) αPS1 subunit (structurally similar to vertebrate laminin-responsive α3, α6, and α7 subunits) dimerizes with an βPS subunit (similar to the vertebrate β1 subunit) to form the laminin-binding PS1 integrin. An αPS2 subunit (similar to vertebrate RGD-dependent α5, α8, αv, and αIIb subunits) also dimerizes with the βPS subunit to form PS2 integrin that binds to molecules with the amino acid triplet RGD (e.g., tiggrin). These PS integrins serve as major cell adhesion molecules during embryogenesis, with PS1 integrin concentrated in ectodermal and endodermal tissues and PS2 integrin being most abundant in mesodermal tissues (Zusman et al., 1990; Bunch et al., 1992; Roote and Zusman, 1996). A role for PS integrins in embryonic neuronal differentiation has been speculated because βPS loss-of-function mutants fail to condense the CNS normally during early larvagenesis and primary neuronal culture from these mutants fail to develop normally on a laminin substrate (Donady and Seecof, 1972; Brown, 1994). The αPS3 subunit, a new member of the PS integrin family that dimerizes with a βPS subunit, has also been detected in the embryonic nervous system (Stark et al., 1997). Both laminin and tiggrin, known ligands for PS1 and PS2 integrins, respectively, are present along the embryonic axon pathways (Montell and Goodman, 1989; Fogerty et al., 1994). Despite all circumstantial evidence that they may be involved in neural development, expression patterns of PS integrins as well as their specific role during neuronal development have not yet been examined.
In this study, we show that PS integrins (αPS1, αPS2, and βPS subunits) are expressed in the embryonic nervous system, starting during the period of axogenesis, and we provide genetic evidence that neuronally expressed integrins are directly involved in axon development.
MATERIALS AND METHODS
Fly stocks. Null alleles for the αPS1 integrin subunit gene multiple edematous wings arey 1 mew M6 f 36a p[ry +t7.2 :newFRT] 18A /FM7c ftz’-lacZ(a null allele) and y 1 mew 498 p[ry +t7.2 :newFRT] 18A /FM7c(a protein null allele); loss-of-function alleles for the αPS2 integrin subunit gene inflated areg 2 if K27E f 36a /FM7c ftz’-lacZ (a null allele) and g 2 if B2 f 36a /FM7c (a hypomorphic allele with an ∼10% protein expression level) (Brabant and Brower, 1993;Brower et al., 1995) (source, K. Stark, Massachusetts Institute of Technology, and Bloomington Fly Stock Center, Bloomington, IN). All of these mutations reach lethality by early larval stages. The genotypes of individual embryos examined were confirmed immunologically using the appropriate PS subunit antibodies, which reveal the presence or absence of particular PS subunits, and/or β-galactosidase antibodies, which detect the FM7c balancer chromosome that carries the marker transgene ftz′-lacZ (see Immunocytochemistry). For “neuron” rescue experiments, wild-type αPS2 gene was supplied to the nervous system in αPS2 null mutants (if K27E) by the use of the GAL4 misexpression system (Brand et al., 1994) that combines a genomic neurotopic enhancer “elav’-GAL4 III” (Sone et al., 1997) (source, E. Suzuki, University of Tokyo) to the GAL4-responsive transgene “UAS-αPS2 wt” (Roote and Zusman, 1996) (source, D. Brower, University of Arizona):if K27E /Y; UAS-αPS2 wt /elav’-GAL4 III. Misexpression of αPS1 and αPS2 was achieved by, respectively,GAL4 C155 /+ (or Y); UAS-αPS1 wt /+ andGAL4 C155 /+ (or Y); UAS-αPS2 wt /+, in whichGAL4 C155, a GAL4 “enhancer trap” line (D. M. Lin et al., 1994) (source, C. Goodman, University of California at Berkeley), targets misexpression to all neurons similar to the “elav’-GAL4 III” line.Canton S strain was used as a wild-type control. In addition, the βPS null mutant line (mys xb87 /Y) (Leptin et al., 1989) (source, K. Stark) was examined as a negative control for the immunocytochemistry data.
In situ hybridization. cDNAs for αPS1 (multiple edematous wings gene), αPS2 (inflated gene), and βPS (myospheroid gene) were subcloned into pBluescript to prepare digoxygenin-labeled antisense and sense (negative control) RNA probes (Bogaert et al., 1987; MacKrell et al., 1988; Wehrli et al., 1993) (source, D. Brower). Wild-type 9–18 hr whole embryos were processed for in situ hybridization and probed with RNA at concentrations of ∼1 ng/ml (βPS and αPS1) and ∼5 ng/ml (αPS2) for 2–3 hr (Broadus and Doe, 1995). The entire set ofin situ hybridization experiments was repeated independently three times to confirm reproducibility of the staining patterns.
Immunocytochemistry. Primary antibodies were as follows: mAb CF.6G11 (βPS subunit; 1:1000 dilution), mAb DK.1A4 (αPS1 subunit; 1:500 dilution), mAb CF.2C7 (αPS2 subunit; 1:500 dilution) (Wilcox et al., 1981; Brower et al., 1984) (source, D. Brower), mAb 1D4 (1:4 dilution) (Grenningloh et al., 1991) (source, C. Goodman), and anti-β-galactosidase (1:5000 dilution) (source, Promega, Madison, WI). Embryos were immunoprocessed as whole embryos or after fillet dissection on glass slides with “minipools” (Chiba et al., 1993). The mutant embryos were analyzed by double-immunolabeling with mAb 1D4 that stains CNS axon fascicles and motoneuron axons and an antibody against the respective integrin subunit and/or β-galactosidase (see Fly stocks). To preserve antigenicities for the PS subunits, we incubated the embryos with the primary antibodies before fixation. Analysis was based on abdominal A2–A7 segments of fillet-dissected preparations.
Embryonic neurons express PS integrins during axogenesis
We examined the expression pattern of αPS1, αPS2, and βPS subunits using in situ hybridization and immunocytochemistry in the embryonic nervous system (see Materials and Methods). All three PS subunit mRNAs are expressed widely in the nervous system (Fig.1). Their expression levels during hours 9–18 of embryogenesis are notably low compared with that in the other tissues that have been studied previously, such as muscles and apodemes (Fig. 1 A–C, asterisks). αPS1 mRNA is detected widely in the CNS at a steady level during hours 13–18 (Fig. 1 A). During this period, axogenesis occurs within the CNS as well as in the periphery. At the ventral midline, cells appear to accumulate slightly higher levels of αPS1 compared with most other cells in the CNS (Fig. 1 A,open arrowheads). The αPS2 mRNA expression pattern differs somewhat from that of αPS1. Specific clusters of cells, one near the midline and a bilateral pair at mediolateral sites, express at relatively high levels of αPS2 in each segment of the CNS (Fig.1 B, arrowheads). The αPS2 expression in the CNS peaks during hours 9–15 of embryogenesis. The βPS mRNA expression pattern partially overlaps with those of αPS1 and αPS2 mRNA in the CNS, with one prominent cluster of cells expressing relatively high levels at the ventral midline (Fig. 1 C,open arrowheads). βPS mRNA expression in the CNS persists through hours 9–18. These in situ hybridization data suggest that the embryonic CNS expresses both PS1 (αPS1/βPS heterodimer) and PS2 (αPS2/βPS heterodimer) integrins during the period of axogenesis.
Consistent with the mRNA data, immunocytochemistry shows that the βPS protein subunit is unambiguously revealed on neuronal cell surfaces during hours 16–18 (Fig.2 A). The major axon fascicles within the CNS, including the longitudinal connectives and the anterior and posterior nerve tracts (Fig. 2 A), as well as the cell surfaces of at least some identified neurons, are labeled with the βPS antibodies (Fig. 2 A,arrowheads). Unfortunately, both αPS1 and αPS2 subunits are below the threshold of detection with available antibodies. However, the detection of the βPS protein subunit on the neuronal cell surfaces suggests that the αPS subunits are likely forming functional heterodimers with the βPS subunit in those cells. These observations with in situ hybridization and immunocytochemistry have led us to conclude that, similar to vertebrate neurons, many Drosophila neurons express relatively low levels of integrin on the neuronal surface during axogenesis.
Loss of integrin disrupts axon guidance
To analyze the role of the PS integrins in developing axons, we examined mutant embryos lacking the functional gene for either the αPS1 or αPS2 subunit. In these mutants, unlike in the βPS null mutants that exhibit grossly abnormal muscle development (Leptin et al., 1989), muscles develop apparently normally up to hour 18 through the major period of embryonic axogenesis. There is little sign that neuronal cell bodies have migrated to incorrect positions, in addition to the fact that the entire nervous system develops relatively normally. On closer examinations, however, both αPS1 and αPS2 loss-of-function mutant alleles exhibit similarly widespread and variable axon guidance defects (Fig.3 B,C). In the CNS, the longitudinal connective normally contains three prominent axon fascicles that are easily visualized by mAb 1D4 (Fig.3 A). Axons in the mutants appear somewhat wiggly (Fig.3 C, circle) or partially disconnected (Fig.3 B, arrows). The results are summarized in Figure4.
In the PNS, one can visualize specific groups of motoneuron axons with higher cellular resolution than is possible in the CNS. Null mutations in αPS1 and αPS2 subunit genes both result in similarly widespread axon pathfinding defects for all five known motoneuron groups, despite apparently normal muscle development (Fig.5 G–L). The axon defects observed can be interpreted as a consequence of failing to turn at choice points and/or invading into neighboring muscle fields (Fig. 5,arrows, circles). These defects are most frequently detected in the SNb group (Fig.4 D,F). It is important to note that the axons in these loss-of-function mutants can extend as far as in wild type and sometimes up to 30 μm beyond their normal stopping points (Fig. 5 J, circle). This suggests that integrin is unlikely to serve simply as a clutch-constituting molecule, on which growth cones depend for the adequate traction needed to extend forward. Another point is that each axon group selects a range of alternative pathways without obvious preferences. It is therefore likely that the loss of integrin leads to losses in the responsiveness of an axon to a large array, rather than a small specific set, of guidance cues. Finally, in general, loss of PS2 integrin (αPS2 null mutation) leads to higher axon guidance errors than does loss of PS1 integrin (αPS1 null mutation). Future analysis is needed to determine specific contributions of these two forms of integrin. On the basis of the current data, we suggest that both the laminin-binding PS1 and RGD-dependent PS2 integrins are necessary for accurate axon guidance.
Axon guidance defects are rescued by supplying the wild-type integrin
To test the direct role of integrin in axon development further, we attempted to rescue the axon defects by using a genomic neurotopic enhancer to drive expression of wild-type integrin gene in the nervous system of the null mutant (see Materials and Methods). We chose to focus on the αPS2 null mutants because they showed more severe PNS defects than did the αPS1 null mutants (Fig.6). The overall expression levels of the αPS2 protein subunit in this neuron rescue experiment were low and hardly detectable via immunocytochemistry, approximately mimicking endogenous expression in the CNS (data not shown). Consistent with a direct role for integrin, guidance defects are primarily rescued in both the CNS longitudinal connectives and the peripheral motor pathways (Figs. 6, 7). This result suggests that axons rely on their own integrin while responding accurately to local guidance cues.
Integrin misexpression results in little growth cone guidance errors
How does neuronal integrin facilitate accurate axon guidance? A simple model is that integrin itself serves as a receptor for specific guidance cues, in much the same way as do a number of cell surface receptors (see Discussion). To test this idea, we expressed integrin ubiquitously in the wild-type CNS so that it would be present in neurons that normally express little or no integrin. We predicted that, if integrin serves as a receptor for specific growth cone guidance cues, many neurons with normally low or no integrin expression would alter their axon pathways. Contrary to our prediction, we found that axons exhibit little pathfinding error in either the CNS or periphery when either the αPS1 or αPS2 gene is misexpressed ubiquitously in the nervous system (Fig. 8). These results, as well as the results from the loss-of-function analysis described above, suggest that integrin is not likely to serve as a specific guidance receptor in vivo.
In this study, we have shown that integrin (αPS1, αPS2, and βPS subunits) is present in the developing Drosophilanervous system and have provided evidence that neuronal integrin is required for accurate axon guidance in vivo.
Neuronal integrin guides axons
Integrin, one of the best-studied classes of cell adhesion/signaling molecules in animals, has been shown to link intercellular signaling systems to a number of intracellular signaling pathways (Parsons, 1996; Schlaepfer and Hunter, 1998). It is expressed dynamically in a variety of cells during development wherein extensive intercellular communication is required. Evidence of potential roles of integrin during axon development is available from in vitroexperiments (Schmidt et al., 1995; Varnum-Finney et al., 1995;Felsenfeld et al., 1996; Shaw et al., 1996; Wu et al., 1996; Grabham and Goldberg, 1997). However, recent mouse knock-out experiments fail to demonstrate the specific in vivo role of integrin during axon development, primarily because of the large number of integrin isoforms and their widespread expression patterns (Hynes, 1996). Therefore, to date, an in vivo demonstration of the roles of integrin in axon guidance has not been available.
Our genetic analysis on the Drosophila embryonic nervous system provides two lines of evidence that neuronal integrin is essential for accurate axon guidance. First, in the loss-of-function mutants for the αPS1 or αPS2 subunit gene, axons in the CNS and the periphery both exhibit widespread guidance errors (see Fig. 4 for summary). In the PNS, with its relatively simple cellular landscape, the miswired axons are seen frequently to extend beyond normal target regions (Fig. 5 J,L). Therefore, although lack of integrin does not reduce general motility of growth cones, the accuracy of their guidance is diminished. Second, genetic rescue reverts the axonal defects toward the wild type (Figs. 6, 7). The fact that this is achieved by resupplying low levels of the wild-type integrin gene specifically to the nervous system of otherwise null mutant embryos strongly suggests that integrin is directly involved in the axon guidance.
Neuronal integrin and its low expression levels
Previous studies on Drosophila PS integrins have focused mostly on their involvement during the development of non-neuronal tissues, including wing epithelia, muscle attachment sites, dorsal vessels, and gut (Leptin et al., 1989; Wilcox et al., 1989; Wilcox, 1990; Zusman et al., 1990; Brown, 1993; Gotwals et al., 1994b; Brower et al., 1995; Fernandes et al., 1996; Stark et al., 1997;Bunch et al., 1998). The primary function of integrin is thought to be that of cell adhesion in these nonmigratory cells. Much less is known about the roles of integrin in the nervous system, where it is expressed at relatively low levels. In vitro work with chick dorsal root ganglion neurons shows that protein kinase C-dependent cytoplasmic signaling is involved in the behavioral modification of growth cones that occurs after their contacting laminin, the major ligand for integrin (Kuhn et al., 1995). This suggests that in axons, with its relatively low expression levels, integrin may primarily serve as a mediator of cell signaling via its specific association with a variety of cytoplasmic proteins. In vivo analysis on the physiological functions of the cytoplasmic domains of the PS integrin subunits in Drosophila have just begun in a variety of tissues (Martin-Bermudo et al., 1997; Li et al., 1998). It would be interesting to extend such analysis and test the roles of the cytoplasmic domains of integrin in the context of axon guidance.
New models for the role of integrin in axon guidance
Although our genetic analysis demonstrates that neuronally expressed integrin is essential for accurate axon guidance, the results are inconsistent with some of the previously proposed models concerning the role of integrin in axon development. One model, known as the clutch model, stems from the in vitro observations that, when a growth cone advances, actin filaments inside filopodia are held down to the substrate (Lin and Forscher, 1995). When applied to integrin, this clutch model proposes that integrin, an integral membrane molecule, serves as a mechanical link between specific extracellular substrates and the cytoskeleton and thus provides the traction necessary for growth cone advancement. Support for this “integrin-as-clutch” model comes from an in vitro study in which integrin activation is suggested to lock integrin to actin filaments in filopodia (Felsenfeld et al., 1996). The model predicts that, when integrin is missing, growth cones will lose much of their traction and will reduce their forward motility. However, our in vivo analysis has revealed little reduction in growth cone motility as a result of genetic deletion of the integrin subunits. Instead, there is an increase in growth cone targeting errors. Thus, a simple clutch model does not seem to adequately explain the role of integrin in axon development.
In the alternative receptor model, neuronal integrin itself is proposed to serve a pivotal role in turning a growth cone by reacting to specifically localized extrinsic cues (Kuhn et al., 1995). This model predicts that, when integrin is lacking, growth cones that normally express integrin will select alternative pathways at the choice points where the ligands of integrin are normally present. Along the same line of logic, if integrin is misexpressed in growth cones that normally do not express it, this will alter the pathfinding of these growth cones in specific ways as well. Our observations suggest that this receptor model is not predictive of the role of integrin in axon guidance. It is noteworthy that, in vitro, integrin is clearly capable of exerting enough biases on a growth cone to turn it toward the source of specific ligands such as laminin (Kuhn et al., 1998). However, whether the natural ligands of integrin are distributed in specific patternsin vivo is still not clear. Available evidence hints that laminin and tiggrin, the known ligands for the PS integrins inDrosophila, are both rather ubiquitously expressed along the entire substrate that the majority of neuronal axons grow over (Montell and Goodman, 1989; Fogerty et al., 1994). Therefore, we favor the view that, in vivo, integrin is unlikely to serve as a specific axon guidance receptor.
What then is the role of integrin in the axon guidance, because its absence does lead to widespread loss of guidance accuracy? In one simple model, integrin functions as an adhesion molecule that controls the speed of growth cone advancement. In this “speed control” model, low levels of integrin are necessary to prevent the axons from extending too fast and missing the chance to interact with guidance cues. In a more elaborate model, integrin serves as an interface that links a specific cue recognition at the surface of the growth cone to internal cytoplasmic and cytoskeletal signaling events. In this “interface” model, integrin cooperates with a number of other growth cone receptors, such as EphR family receptor tyrosine kinases, DCC-type netrin receptors, receptor-type phosphotyrosine phosphatases, Fasciclin III, Connectin, and other unknown receptors (Speicher et al., 1998; Treubert and Brummendorf, 1998). Activation of any of these specific growth cone receptors is coupled to integrin-mediated intracellular signaling systems. The model predicts that integrin and associated molecules are rapidly recruited to, and/or activated at, particular filopodia that have contacted a specific target or guidepost cell. It also anticipates that mutations that delete only one of the many different growth cone receptors that are interfaced by integrin do not always produce pronounced axon guidance errors. On the other hand, deleting integrin would cause widespread axon defects, as observed in this study. Integrin, with its extensive opportunity to work with a large array of molecules (Clark and Brugge, 1995; Schlaepfer and Hunter, 1998), seems to be in an ideal position to signal-amplify a local event of minimal amplitude and to generate a bias large enough to steer a growth cone with accuracy.
This work was supported by National Institutes of Health Grant NS35049, National Science Foundation Grant IBN-95-14531, and the Lucille P. Markey Charitable Trust (A.C.). We thank Dan Brower and Mike Graner (University of Arizona), Karen Stark (Massachusetts Institute of Technology), Corey Goodman (University of California at Berkeley), and Emiko Suzuki (University of Tokyo) for generous gifts of reagents. We also thank Hiroyuki Kose (National Institute of Genetics, Japan) and Yasumitsu Takagi (Tsukuba Research Consortium, Japan) for discussion and Anna Huttenlocher, Steven Kaufman, Xiaomao Zhu, and the current members of the Chiba Lab (University of Illinois) for comments on this manuscript.
Correspondence should be addressed to Dr. Akira Chiba, B605 Chemical and Life Science Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801.