Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 28 (2005) 141 – 152

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila Lars V. Kristiansen,a,b,c Emma Velasquez,a Susana Romani,a,1 Sigrid Baars,a Vladimir Berezin,b Elisabeth Bock,b Michael Hortsch,c,* and Luis Garcia-Alonsoa,* a

Instituto de Neurociencias CSIC-UMH, Universidad Miguel Hernandez, Sant Joan d’Alacant, 03550 Spain Institute of Molecular Pathology, Panum Institute, University of Copenhagen, Copenhagen N, DK-2200 Denmark c Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA b

Received 1 July 2004; revised 30 August 2004; accepted 2 September 2004 Available online 27 October 2004

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. We have studied the functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-offunction (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, our findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM. D 2004 Elsevier Inc. All rights reserved.

Introduction During axon pathfinding, multiple signals and receptors function coordinately in guiding axons through a complex cellular environment. Several distinct families of immunoglobulin * Corresponding authors. Luis Garcia-Alonso is to be contacted at Instituto de Neurociencias CSIC-UMH, Universidad Miguel Hernandez, Sant Joan d’Alacant, 03550 Spain. Fax: +34 965 91 95 61. Michael Hortsch, Department of Cell and Developmental Biology, University of Michigan, 1301 Cathrine Street, Ann Arbor, MI 48109. Fax: +1 734 763 1166. E-mail addresses: [email protected] (L. Garcia-Alonso)8 [email protected] (M. Hortsch). 1 Present address: MRC Centre for Developmental Neurobiology, King’s College, London SE1 1UL, United Kingdom. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.09.003

cell adhesion molecules (CAMs), such as L1- and NCAM-type proteins, developed early during metazoan evolution and are involved in axon extension, guidance, and fasciculation during nervous system development in vertebrates and invertebrates. In all these organisms, L1- and NCAM-type proteins function as homo- and heterophilic adhesion molecules and couple highly specific recognition events between axons and substrates to the activation of neuronal tyrosine kinase signaling pathways to promote neurite extension and axonal guidance (Doherty et al., 1995; Forni et al., 2004; Panicker et al., 2003; Williams et al., 1994). Induction of neurite outgrowth by Ig-CAMs is mediated by two complementing tyrosine kinase signaling pathways (Kiryushko et al., 2004; Niethammer et al., 2002). In Drosophila, mutants lacking the L1-type Neuroglian (Nrg) or the NCAM-type Fasciclin II (FasII) protein display a variety of different, partially penetrant axon guidance phenotypes (GarciaAlonso et al., 2000; Hall and Bieber, 1997; Lin et al., 1994). In vertebrates, L1-CAM knockout mice exhibit abnormal axonal guidance in the corticospinal tract, the corpus callosum, and other developmental central nervous system alterations with partial expressivity (Cohen et al., 1997; Demyanenko et al., 1999). Independent of its homophilic interaction, along with Neuropilin, L1-CAM functions as a specific coreceptor for Semaphorin 3A (Castellani et al., 2000). Emphasizing the importance of these L1-specific, heterophilic interactions, mutant mice with L1-CAM protein lacking its homophilic binding ability, but maintaining its Neuropilin/Semaphorin 3A interaction capacity, do not exhibit the axonal development phenotype (Itoh et al., 2004). The remaining aspects of the phenotype are highly variable and depend on the genetic background. This indicates that other gene products, such as paralogous and functional redundant proteins, appear to strongly influence the expression of the L1-CAM loss-of-function (LOF) phenotypic traits that are controlled by homophilic interactions. In both vertebrate and invertebrate species, the expression patterns of L1- and NCAM-type proteins are partially over-

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lapping (Bieber et al., 1989; Grenningloh et al., 1991; Lustig et al., 2001; Moscoso and Sanes, 1995). Similar to L1-type proteins, NCAM-type proteins mediate homophilic binding, which induces neuronal fibroblast growth factor receptor (FGFR) activity (Cavallaro et al., 2001; Forni et al., 2004; Rønn et al., 1999; Williams et al., 1994). Interestingly, several in vitro experiments indicate that L1-CAM and NCAM appear to cooperate during neurite outgrowth (Kadmon et al., 1990; Kristiansen et al., 1999). In addition, vertebrate NCAM-type proteins are also involved in many unique functions that are independent of their homophilic binding. One example for such an NCAM-specific activity is its function as a coreceptor for a Neurotrophin, GDNF (Paratcha et al., 2003), demonstrating that NCAM is involved in both long range and contact-mediated axon guidance cues. These findings suggest that different types of adhesive molecules do mediate some of their biological activities through specific signaling cascades, while at the same time sharing other signaling pathways. Therefore, it appears likely that structurally different CAMs cooperate in certain neurons during specific developmental processes and that they perform partially redundant functions. We used the postembryonic development of the Drosophila peripheral sensory nervous system as a model to investigate the specific requirements for Drosophila Nrg and the NCAM-type protein FasII during sensory axon guidance. nrg LOF conditions in the Drosophila ocellar sensory system (OSS) result in an axonal misguidance phenotype for both ocellar pioneer (OP) and bristle mechanosensory (BM) neurons (Garcia-Alonso et al., 2000). As demonstrated earlier, this neuronal phenotype can be suppressed by increasing the activity of EGF and FGF receptors. Testing for the rescue of the nrg LOF condition in the OSS, we now show that the neural protein isoforms of Nrg and FasII perform an interchangeable function in OP neurons, which normally coexpress both proteins. In contrast, in OSS BM neurons, which do not express FasII, the ectopic expression of FasII protein enhances the nrg LOF axon misguidance phenotype. This cell-specific, redundant function, which is also uncovered by nrg fasII double mutant analysis, does not extend to the nonneural isoform of Nrg. In addition, when expressed in Drosophila, the corresponding vertebrate L1- and NCAM-type proteins display the same cellular and functional specificities as their Drosophila homologs. Finally, we show that vertebrate L1- and NCAM-type proteins produce the same gain-of-function (GOF) phenotype in the sensory nervous system of the developing wing as their Drosophila counterparts. We previously demonstrated that this GOF phenotype is mediated by the homophilic adhesion-dependent activation of endogenous Drosophila epidermal growth factor receptors (EGFR) (Islam et al., 2004). We now expand this finding to FGFR signaling. Our results show that in both the Drosophila wing and the ocellar sensory nervous system, L1- and NCAM-type CAMs activate the same neuronal signaling pathways, which integrate different adhesive interactions and translate them into a coordinated axonal growth and guidance response. Our complementing LOF and GOF analyses indicate that, under in vivo conditions, members from both CAM families have conserved a redundant functional specificity over a long evolutionary time period, indicating that this may be required for ensuring efficient and reliable axonal guidance during nervous system development.

Results Neuroglian180 and transmembrane-Fasciclin II are coexpressed in specific axons of the Drosophila ocellar sensory system In the nervous systems of animals from different phyla, L1- and NCAM-type proteins are expressed in distinct but overlapping patterns. In insects, which only have one L1-type gene, Nrg is widely distributed, while FasII shows a restricted pattern of expression (Bieber et al., 1989; Grenningloh et al., 1991; Harrelson and Goodman, 1988; Wright and Copenhaver, 2001). The nrg gene generates two protein isoforms by differential splicing, Nrg180 and Nrg167 (Hortsch et al., 1990). These isoforms only differ by the presence of an additional 63 amino acid residues at the carboxyterminal end of the neuron-specific Nrg180 isoform. The Nrg167 isoform is expressed in epithelia and many other nonneuronal cells (Hortsch et al., 1990). In contrast, the Nrg180 isoform is specifically expressed in neurons, where the generation of its mRNA splice product is under the control of the elav gene product (Lisbin et al., 2001). Similarly, Drosophila has only one NCAM-type gene, which gives rise to three major protein isoforms, a GPI-membrane anchored isoform, FasIIGPI, and two transmembrane isoforms, FasIIPEST+ and FasIIPEST . The two transmembrane FasII isoforms differ by the presence or absence of a PEST polypeptide motif in their respective cytoplasmic domains. In the ocellar sensory system of Drosophila, OP axons project directly away from the epithelial surface towards the brain. In contrast, the neighboring BM axons follow the epithelial surface towards a choice point near the antenna, where they detach and project towards a different brain region (Figs. 1A and G). The basis for this differential choice of projection trajectory resides in the different interaction of OP and BM axons with the epithelium (Garcia-Alonso et al., 1996). At the early prepupal stages of metamorphosis (before head eversion), the extending OP axons grow detached from the epithelium, while the BM axons extend attached to it (Fig. 1G). At this early stage, no glia cells are involved in the guidance of OP and BM axons (Garcia-Alonso et al., 1996). To analyze the requirements for Nrg and FasII during axon guidance in the OSS, we first had to compare the normal expression pattern of both molecules in OP and BM neurons. As shown in Fig. 1, the neuron-specific isoform Nrg180 is expressed by both OP and BM axons (Figs. 1B–C), while the nonneural isoform Nrg167 is expressed by the epithelium (Garcia-Alonso et al., 2000). In contrast, expression of the transmembrane FasII isoforms is restricted to OP axons, where it is coexpressed with Nrg180 (Figs. 1D–F). This coexpression of Nrg180 and FasII during OP axon guidance could either reflect simultaneous independent requirements for specific functions of each molecule or indicate a cooperative and possibly redundant function. Due to the differential expression of Nrg and FasII and the two different projection pathways of OP and BM axons, the Drosophila OSS offers a good in vivo model for analyzing the differential requirements for L1and NCAM-type proteins and their respective molecular isoforms in neurons. The Nrg requirement for OSS axon guidance is specifically provided by the neural Nrg180 isoform Nrg is required for axon guidance in the embryonic and adult peripheral nervous system (PNS) (Garcia-Alonso et al., 2000; Hall and Bieber, 1997). Previous work has shown that the requirement

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Fig. 1. Specific isoforms of Nrg and FasII are differentially expressed in the OSS. (A) A dorsal view of the normal pattern of sensory axon projections in the pupal OSS. Black arrow points to OP cell bodies and black arrowhead to BM organs (including both the external and the internal bristle cells). The OP nerve projects away from the epidermal surface, and only the three clusters of OP cell bodies are in focus. Open arrowhead points to BM axons projecting along the epidermal surface. The inset displays the corresponding area in the adult head. (B) A dorsal view showing that the neural-specific Nrg180 isoform is expressed by both OP (black arrow) and BM (open arrowhead) neurons (but not by the external bristle cells). (C) A posterior coronal view showing the neural-specific Nrg180 isoform expression in OP cell bodies (black arrow) and OP axons (open arrow). (D) At the prepupal stage, before head eversion, FasII is expressed by OP cell bodies (black arrow) and their axons (open arrow). Expression of this isoform is also evident in trachea (the two tubular structures marked with asterisks). Blue nuclei correspond to lacZ expression from the insertion A101 in neurons and glia (only the lateral OP nuclei near the black arrowhead and the medial OP nuclei close to the middle of the tract are present along the nerve). (E) Dorsal view of the pupal head stained for FasII expression. Lack of signal in BM axons as well as in OP cell bodies is evident. Open arrow points to OP axons. (F) The same pupal stage as in E but in a posterior coronal view. The open arrow points to the strong FasII signal in the OP nerve (note the absence in OP cell bodies at the epidermal layer). (G) Scheme of the head eversion process from a lateral view showing coexpression of the neural-specific Nrg180 and FasII in OP axons and specific CNS pathways (orange color), Nrg167 expression in epidermis (blue color), and neural Nrg180 in BM neurons and CNS without FasII (green). Panel A is stained with MAb 22C10. Panels B and C are stained with MAb BP104 (anti-Nrg180). Bottom three panels (D–F) are stained with MAb 1D4 (anti-transmembrane FasII). Bar = 0.1 mm for A, B, D, and E, and 0.05 mm for C and F.

for Nrg during adult sensory axon guidance is connected to its function to promote FGF and EGF receptor activity (GarciaAlonso et al., 2000; Islam et al., 2004). In the OSS, OP and BM axons normally express the neural-specific Nrg180 isoform (Figs. 1B–C), while the epithelium expresses the nonneural Nrg167 isoform (not shown). Nrg180Nrg180 axon–axon interactions govern the fasciculation of OP axons and their detachment from the epithelium, a process that can fail in nrg mutant animals (Fig. 2A) (Garcia-Alonso et al., 2000). In contrast, BM axons extend on the epithelium by Nrg180–Nrg167 axon–epithelium interactions. In nrg 3 mutants, BM axons sometimes detach from the epithelium, indicating that the epithelial expression of Nrg167 is important for their guidance (Garcia-Alonso et al., 2000).

In this study, we used the GAL4/UAS technique (Brand and Perrimon, 1993) to direct the expression of different Nrg isoforms during postembryonic sensory axon guidance. Since expression of Nrg is required in both OP and BM axons, as well as in the epithelium, we used the GAL4 driver line MS1075, which expresses GAL4 in epithelium and neurons of the OSS (GarciaAlonso et al., 2000), to direct specific expression of the Nrg180 or Nrg167 isoforms in an nrg mutant background. The use of the nrg 3 thermo-sensitive mutation, which generates a null Nrg condition at the restrictive temperature, enabled us to separate the axon guidance function from an earlier requirement for Nrg during neuron pattern formation (Garcia-Alonso et al., 2000). The MS1075-driven expression of neural Nrg180 in the OSS of control

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Fig. 2. OSS axonal requirements for Nrg and FasII isoforms. (A) Typical phenotype of a nrg 3 individual raised at the restrictive temperature (298C). Open arrows point to OP axons ectopically projecting along the epidermis. Open arrowhead points to a stalled BM axon. (B) The Nrg requirement in OSS axons cannot be provided by the nonneural Nrg167 isoform. Expression of Nrg167 in a nrg 3 individual causes the enhancement of the OSS axon alteration expressivity. Open arrows point to several OP axonal fascicles in the epidermis. In addition, other OP axons are tangled between the OP cell bodies. (C) Normal axonal pattern in a fasII e76 mutant. (D) Abnormal OP projection inside the head in the FasI overexpression condition. The OP fascicle has grown detached from the epidermis (note that the epidermis with the BM axons is out of focus) but projects to an ectopic site near the brain. Bar = 0.1 mm. All panels show MAb 22C10 stainings.

individuals causes an overexpression of this isoform in OP and BM neurons and its ectopic expression in the epidermis. This mis- and overexpression condition causes no phenotype in BM axons (0% of 98 heads analyzed at 298C), and only occasionally (8% of 98 heads analyzed at 298C), some OP axons erroneously extended on the epidermis. The MS1075-driven expression of neural Nrg180 in the OSS of nrg 3 mutant individuals restores its expression in OSS neurons (OP and BM) and causes its ectopic expression in the epithelium. This condition rescues both the penetrance (Table 1) and the expressivity of the OP and BM axon misguidance defects in nrg 3 mutant animals. In contrast to Nrg180, the MS1075-driven expression of Nrg167 causes its ectopic expression in OP and BM neurons and its overexpression in the epidermis. This condition displays some weak alterations in OSS axon guidance (8% of the 50 heads analyzed at 298C had some OP, and 14% had some BM axon defects). MS1075-driven expression of Nrg167 in nrg 3 -mutant individuals, which restores the expression of this isoform in the epithelium and causes its ectopic expression in OSS neurons, shows a synergistic increase in the expressivity of OP axon alterations (i.e., axonal alterations were much more severe in those individuals that displayed the phenotype) (Fig. 2B). Although the enhancement of the phenotypic penetrance does not reach a statistically significant level (Table 1), the result clearly indicates

the inability of the Nrg167 isoform to mediate OSS axon guidance on its own. Therefore, only the neuronal Nrg180 isoform can provide full Nrg functionality in OP and BM axons. In addition, Nrg180 is able to substitute for the function of Nrg167 in the epithelium during BM axon guidance. These results reveal a high degree of specificity for the neuronal Nrg isoform during OSS axon guidance. Nrg180 and FasIIPEST+ display neuron type-specific redundancy and antiredundancy during OSS axon guidance The complete removal of the Nrg function causes an axonal phenotype with partial penetrance and expressivity in both the embryonic nervous system (Hall and Bieber, 1997) and the ocellar sensory system (Garcia-Alonso et al., 2000). This suggests the existence of other molecules with redundant capabilities to carry out the same function. In the Drosophila CNS and PNS, FasIIPEST+ is expressed in subsets of axon fascicles (Grenningloh et al., 1991). As presented in Figs. 1D–F, this FasII isoform is normally coexpressed with Nrg180 in OP axons, but it is not expressed in the neighboring BM axons or in the epidermis. In the next set of experiments, we tested the possibility that FasII may function redundantly with Nrg during OP axon guidance.

L.V. Kristiansen et al. / Mol. Cell. Neurosci. 28 (2005) 141–152 Table 1 Rescue of Nrg OSS axon guidance requirements by specific Nrg and FasII protein isoforms OP axon alterations

BM axon alterations

nrg 3

21.5 43/200

30.0 60/200

nrg 3 + Nrg 180

8.3A 14/168 P = 0.0005

2.4A 4/168 P = 0.0001

nrg 3 + Nrg 167

46.1 6/13 P = 0.08

53.8 7/13 P = 0.12

nrg 3 + FasII PEST+

9.8A 6/61 P = 0.04

68.9z 42/61 P = 0.0001

nrg 3 + FasI

58.3 7/12 P = 0.083

33.3 4/12 P = 0.756

Penetrances (percentage of individuals displaying abnormalities) of ocellar pioneer (OP) and bristle mechanosensory (BM) axonal alterations in nrg 3 mutant animals combined with different Nrg and FasIIPEST+ overexpression conditions. Statistically significant ( P b 0.05) suppression (A) or enhancement (z) is marked by bold typeface. The statistical significance value ( P) was calculated comparing the penetrances of the different nrg 3 ;UAS-cDNA/ MS1075 combinations with the nrg 3 ;MS1075/+ controls (all controls for Drosophila or vertebrate cDNAs showed similar penetrance values and were pooled).

Null conditions for fasII (fasII eB112 allele) are embryonic lethal, and fasII eB112 mosaic individuals lack OSS organs (Garcia-Alonso et al., 1995). This precludes a straightforward analysis of the FasII axon guidance requirement in the OSS. However, in the fasII hypomorphic combinations fasII eB112 /fasII e76 and fasII e76 , FasII protein levels are reduced to 5% and 10%, respectively (Grenningloh et al., 1991), which maintains viability and results in only a partial loss of OP and BM neurons. In both fasII e76 and fasII eB112 / fasII e76 individuals, we did not find any OP axon alterations and only some sporadic single BM axon defects (Fig. 2C). These results indicate that a putative FasII requirement for OP axon guidance is less stringent than the corresponding requirement for FasII during OSS neurogenesis. As previously argued for Nrg, this situation would be consistent with the presence of other molecules operating in a redundant fashion along with FasII during OP axon guidance. To test whether FasII functions redundantly with Nrg180 during OP axon guidance, we first studied the effect of a reduction in the gene dosage of FasII on the nrg 3 phenotype (in nrg 3 /fasII eB112 nrg 3 individuals at the restrictive temperature). Heterozygous fasII eB112 /+ flies have 50% of the normal amount of FasII protein (Grenningloh et al., 1991) and display a wild-type phenotype. However, if Nrg and FasII share a common role during OP axon guidance, this condition should enhance the nrg 3 OP axon alterations. As shown in Fig. 3, the protein null fasII eB112 allele acts as a dominant enhancer of the nrg 3 defects in OP axons, where both Nrg180 and FasII are normally coexpressed. However, as expected, it does not modify the axonal guidance defects of BM axons. These results suggest that the functional interaction of FasII and Nrg180 in OP axons is not hierarchical, as in this case, no enhancement of the null nrg 3 phenotype would be expected. Furthermore, the enhancement of the nrg 3 phenotype by a dosage

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reduction of the fasII gene is consistent with the existence of either a functional redundancy between Nrg180 and FasII or a cooperation between specific functions in each protein during OP axon guidance. To distinguish between these two possibilities, we tested whether UAS-directed increased expression of the FasIIPEST+ isoform in the OSS compensates for Nrg180 LOF conditions during OP axon development. As for Nrg, we used the MS1075-GAL4 driver line to induce overexpression of FasIIPEST+ in the OSS. The MS1075driven overexpression of FasIIPEST+ protein causes only a sporadic phenotype (10% of the 40 heads analyzed at 298C had some OP and 15% some BM axon alterations). As an additional control for specificity, we studied the overexpression condition for another Drosophila CAM, Fasciclin I (FasI), which is not a member of the Ig-domain superfamily (Zinn et al., 1988). FasI is also expressed during the development of sensory organs in larval imaginal discs and pupae (Whitlock, 1993). Mutant individuals lacking FasI (fasI TE ) display a normal axon guidance of sensory axons in the wing (Whitlock, 1993) and in the OSS (data not shown). In contrast to FasIIPEST+, the MS1075-driven overexpression of FasI caused frequent and stronger alterations in OSS axon guidance (52% and 42% for OP and BM axons, respectively, in 19 individuals; Fig. 2D), revealing that its overexpression is detrimental during OSS axon guidance. Most of these alterations consisted of OP axons failing to enter the brain at the correct choice point. This phenotype is very similar to a subtype of OP axon alterations found in the nrg 3 null condition (Garcia-Alonso et al., 2000). Therefore, these results indicate a different functional capacity of FasI and FasIIPEST+ during OSS development. Using the MS1075 driver line, we tested whether the overexpression of FasI or FasIIPEST+ can rescue the nrg 3 phenotype at the restrictive temperature. FasIIPEST+ expression rescued OP axon alterations to a similar extent as the neural Nrg180 isoform (Table 1). In contrast, the expression of the FasIIPEST+ isoform in BM axons and the epidermis did not rescue the nrg 3 BM axonal alterations, but rather enhanced them synergistically (Table 1). As expected, the expression of FasI in an nrg 3 null background did not modify the nrg LOF axonal alterations and caused a phenotype that

Fig. 3. FasII cooperates with Nrg180 during OP axon guidance. A reduction of 50% in the normal dose of the fasII gene in nrg 3 individuals causes a significant enhancement of the OP axon alterations but not of BM axon alterations. White bars correspond to nrg 3 and black bars to fasII eB112 nrg 3 / nrg 3 pupae. The ordinate shows the penetrance (percentage of individuals displaying abnormalities) value of OSS axon (OP or BM) alterations. N values in abscissa indicate the number of analyzed pupal heads. Significance levels: **P = 0.0001.

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reflects a summation of the fasI GOF and the nrg 3 LOF condition (Table 1). This observation is consistent with the idea that FasI and Nrg have different functional capacities during OSS guidance. Together with the fasII LOF analysis described above, these results indicate that the requirement for Nrg180 during OP axon guidance can be satisfied by an increased amount of FasII protein. In addition, FasIIPEST+ expression in BM axons and epidermis in a Nrg null background specifically perturbs BM axonal guidance, indicating an antiredundant (i.e., acting in opposition to) (Krakauer and Plotkin, 2002) capacity to Nrg in these axons. In summary, the nrg LOF rescue analysis indicates a common functional specificity of Nrg180 and FasIIPEST+ in those axons where they are normally coexpressed (OP), but an opposing functional capacity in BM axons, which do not normally express FasIIPEST+. Vertebrate L1-type proteins display a similar capacity as the Nrg180 isoform to sustain OP axon guidance in Drosophila In contrast to the situation for insect L1- and NCAM-type proteins, L1-type proteins are expressed in a more restricted pattern in vertebrates than NCAM. Nevertheless, in both types of animals, a general pattern of coexpression has been maintained. As in the case of Drosophila Neuroglian, two splice isoforms exist for vetebrate L1-CAMs, one specifically expressed in neuronal cells (L1CAMRSLE+) and the other in nonneuronal cells (L1-CAMRSLE ) (Jouet et al., 1995; Miura et al., 1991). However, in contrast to Drosophila Neuroglian, the vertebrate neuronal L1-CAM isoform differs from the nonneuronal isoform by the inclusion of two small exons, which encode two amino acid motifs, YEGHHV and RSLE, and are inserted into the extracellular and the cytoplasmic protein domain, respectively. De Angelis et al. (2001) reported that in an in vitro assay, the L1-CAMRSLE isoform exhibits a considerably lower homophilic binding activity than the RSLE+ isoform. Since both alternatively spliced amino acid motifs do not exist in Drosophila Neuroglian, they are not expected to influence the expression of vertebrate L1-CAM proteins in Drosophila. To test whether vertebrate L1-type proteins have maintained a similar functional specificity as Nrg, we generated UAS transformant lines for both vertebrate L1-CAM isoforms, L1CAMRSLE+from mouse and L1-CAMRSLE from human, as well as for rat Nr-CAM (another vertebrate member of the L1 family) (Grumet et al., 1991). These transgenic lines were used to express vertebrate L1-type proteins in an nrg 3 background. For these rescue experiments, we choose the human UAS-L1-CAM RSLE line because its expression in an nrg + wild-type background induced fewer phenotypic alterations when compared with the mouse UASL1-CAM RSLE+ transformant. A high level of ectopic expressioninduced alterations could mask a potential rescue of the nrg 3 axonal phenotype. The L1-CAMRSLE transformant line did not cause any ectopic expression alterations in the OSS (0% for both OP and BM axons in 14 heads) and only a weak GOF phenotype in the wing (see results below), consistent with its lower homophilic binding activity. The MS1075-driver line was used to direct the ectopic expression of these transgenes in neurons and epidermis of the Drosophila OSS. The expression of human L1-CAMRSLE or rat Nr-CAM in nrg 3 mutant individuals rescued the axonal defects in OP axons to an extent similar to Nrg180 (Table 2). Therefore, it appears that both vertebrate L1-type molecules can correctly interact with the endogenous effector molecules that mediate Nrg function in these axons. Although both human L1-CAMRSLE and rat Nr-CAM expression also slightly reduced the level of BM axon defects, this

reduction was statistically not significant. Therefore, the functional specificity of vertebrate L1-type proteins within Drosophila resembles more closely that of Nrg180 than that of the nonneural Nrg167 isoform. Human NCAM140 displays the same Nrg180-related functional redundancy and antiredundancy as Drosophila FasIIPEST+ Since Nrg180 and human L1-CAM share a similar specificity to operate in OP axons, we tested whether the observed redundant specificity of Nrg180 and FasIIPEST+ is also conserved in the vertebrate NCAM molecule. The human NCAM140 isoform is normally expressed throughout development of the nervous system and therefore might perform similar functions as FasII during axon guidance. As an additional control for specificity, we also expressed rat CD2 in the Drosophila OSS. CD2 represents another adhesive protein from a different Ig-domain gene family, which is expressed in the vertebrate immune system (van der Merwe, 1999). The MS1075-driven expression of human NCAM140 or CD2 directs their ectopic expression in both types of neurons and the epithelium of the OSS. In a wild-type background, expression of human NCAM140 only causes minor OSS axon guidance alterations in a few individuals (9% and 14% of 22 individuals analyzed displayed some OP and BM axons defects, respectively), and rat CD2 exhibits no OSS phenotype at all. The analysis of human NCAM140 expression in nrg 3 individuals shows that it displays the same capacity as FasIIPEST+ to substitute for Nrg180 in OP neurons and to disturb BM axon guidance (Table 2). In contrast, rat CD2 expression in nrg 3 individuals does not affect OP or BM axon alterations in any way (Table 2). Therefore, the experimental results presented here demonstrate that L1- and NCAM-type proteins have conserved a common functional specificity in certain neurons, which normally coexpress these molecules. Vertebrate and Drosophila L1- and NCAM-type proteins produce the same RTK-dependent GOF phenotype in the Drosophila wing In Drosophila, Nrg function during axon guidance in the OSS involves the activation of FGF and EGF receptors (Garcia-Alonso et al., 2000). Similarly, in vertebrate in vitro systems, L1-CAM and NCAM promote FGFR activity and neurite outgrowth (Cavallaro et al., 2001; Williams et al., 1994). In Drosophila S2 cells, homophilic, L1-CAM-mediated adhesion triggers human EGF receptor kinase activity (Islam et al., 2004). This is a specific effect of human L1-CAM controlling human EGFR, since Drosophila FasI homophilic binding does not activate human EGFR (Islam et al., 2004). Similarly, Drosophila Nrg and FasII homophilic interactions promote neurite extension in vitro by activating Drosophila FGFR (Forni et al., 2004). We therefore tested whether L1- and NCAM-type proteins from vertebrates and invertebrates share a conserved functional specificity in controlling FGFR and/or EGFR activity in vivo. In a previous report, we demonstrated that the overexpression of different Nrg isoforms in the developing Drosophila wing induces sensory axon guidance alterations, which can be partially rescued by a genetic reduction of EGFR activity (Islam et al., 2004). We therefore analyzed GOF conditions in the wing for Nrg167, Nrg180, NrgGPI, L1-CAM (RSLE+ from mouse and RSLE from human), Nr-CAM (from rat), the two natural transmembrane isoforms of FasII (FasIIPEST+ and FasIIPEST ), and the NCAM140 isoform (from human and rat). As controls, we studied the corresponding GOF

L.V. Kristiansen et al. / Mol. Cell. Neurosci. 28 (2005) 141–152 Table 2 Rescue of Nrg OSS axon guidance requirements by L1- and NCAM-type protein isoforms OP axon alterations

BM axon alterations

nrg 3

21.5 43/200

30.0 60/200

nrg 3 + humanL1-CAM RSLE

0.0A 0/20 P = 0.016

20.0 4/20 P = 0.44

nrg 3 + ratNr-CAM

4.0A 2/50 P = 0.003

20.0 10/50 P = 0.217

nrg 3 + humanNCAM 140

1.7A 1/60 P = 0.0001

55.0z 33/60 P = 0.0007

nrg 3 + ratCD2

26.5 9/34 P = 0.509

35.3 12/34 P = 0.550

Penetrances (percentage of individuals displaying abnormalities) of ocellar pioneer (OP) and bristle mechanosensory (BM) axonal alterations in nrg 3 mutant animals combined with different L1- and NCAM-type protein overexpression conditions. Statistically significant ( P b 0.05) suppression (A) or enhancement (z) is marked by bold typeface. The statistical significance value ( P) was calculated comparing the penetrances of the different nrg3;UAS-cDNA/MS1075 combinations with the nrg 3 ;MS1075/+ controls (all controls for Drosophila or vertebrate cDNAs showed similar penetrance values and were pooled).

conditions for Drosophila FasI and rat CD2. The MS1075 (and also MS1096-)-driven overexpressions of these L1- or NCAM-type proteins all induce a similar range of axon guidance alterations in the wing, but with different expressivities (Fig. 4). The strength of the GOF phenotype directly correlated with the amount of protein expressed from these transgenes (data not shown). Interestingly, FasI GOF conditions cause a similar phenotype, whereas rat CD2 overexpression does not (Table 3). As for the OSS, human L1CAMRSLE caused a weak GOF phenotype (7% penetrance for wing axon alterations at 298C). In contrast, mouse L1-CAMRSLE+ caused much stronger alterations (81% at 258C). It appears that these Drosophila adhesion proteins and the vertebrate L1- and NCAM-type molecules share a functional capacity that is not present in the vertebrate CD2 protein, which is also a member of the Ig-domain superfamily. Experiments using the neuronal-specific elav-GAL4 driver to express NCAM140, Nrg167, or NrgGPI did not result in a GOF misguidance phenotype. This suggests that an increased number of homophilic interactions between the opposing cell membranes of axons and the wing epithelium is a requirement for producing the axon alterations in the wing. Indeed, at early stages of axon extension, numerous ectopic contacts between axons and epithelium can be observed in MS1075-driven GOF conditions (Fig. 4G). These results support the model that pioneer axons in the Drosophila wing are guided by interactions with the wing epithelium (Palka, 1986). Similar to the situation in the OSS, wing nerve pathways are first pioneered by sensory axons, which are then followed by migrating glial cells (Giangrande, 1994). Since the penetrance and expressivity are variable between different insertion lines of the same UAS construct, we choose a set of lines with the highest penetrance

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for the following genetic interaction studies. These included Nrg 180 , Nrg 167 , Nrg GPI , mouse L1-CAM RSLE+ , Nr-CAM, FasIIPEST (which produced a higher phenotypic penetrance than FasIIPEST+), human NCAM140, and FasI. In principle, the GOF phenotype could simply be explained by an alteration of the adhesive axon–epithelium interaction. However, the Nrg167 and NrgGPI GOF phenotype in the wing is caused in part by an overactivation of EGFR function (Islam et al., 2004). In addition, the loss of FGFR or EGFR function causes a similar phenotype during OSS axon extension as the nrg 3 null condition, which itself can be rescued by GOF conditions of either the FGFR or the EGFR (Garcia-Alonso et al., 2000). Thus, the combined LOF and GOF analyses have demonstrated that, during PNS axon guidance, Nrg functions through EGF and FGF receptor tyrosine kinases. Indeed, the expression of a constitutively activated form of the EGFR causes a similar GOF phenotype in the wing as the GOF conditions for L1and NCAM-type proteins (Fig. 4B). Therefore, we tested whether in all cases the observed GOF misguidance phenotype is mediated by endogenous Drosophila EGFR and FGFR tyrosine kinase activity. This analysis was performed by inducing the overexpression of different L1- or NCAM-type or of control proteins in a genetic background with half the normal gene dose of either one or both EGF and FGF receptors. This was accomplished by generating simple and double heterozygous conditions for the Drosophila EGFR gene torpedo (top) and the FGFR gene heartless (htl). All L1- and NCAM-type GOF conditions tested were sensitive to a reduction in the endogenous dosage of these RTKs (Table 3), indicating that similar to the Drosophila CAMs, the homologous vertebrate proteins are able to function through endogenous Drosophila EGF and FGF receptor activities. L1- and NCAM-type protein GOF conditions were suppressed in both penetrance and expressivity by a separate or simultaneous reduction of both RTKs. In contrast, the penetrance of the FasI GOF phenotype was not significantly suppressed by the simultaneous reduction in the EGFR and FGFR dosage (Table 3). We observed a reduction of its expressivity (Figs. 4G, H), including a reduction in the number of ectopic axon–epithelium contacts at early stages of axonal extension (Fig. 4H). This suggests that RTKs are also involved in the generation or the stabilization of the filopodial contacts, which are mediated by FasI homophilic binding. Previously, we demonstrated that the expression of NrgGPI in the wing also produces the EGFR-dependent GOF phenotype in a Nrg null background (Islam et al., 2004). Therefore, the extracellular domain of Nrg is sufficient to induce the observed GOF axonal misguidance phenotype. Our genetic analysis suggests that both L1- and NCAM-type proteins contain not only the capacity for homophilic binding in their extracellular domain, but also the ability to activate RTKs. It remains to be elucidated whether the functional convergence of multiple CAMs on the EGF and FGF receptor signaling pathway always represents a direct physical interaction as has been shown for Nrg and EGFR (Islam et al., 2003). For FasI, the mechanism might well be indirect and perhaps similar to the situation found in the sea urchin embryo, where the FasI-related protein EBP associates with the EGF-related peptide EGIP-D (Hirate et al., 1999).

Discussion In this study, we present an analysis of the requirements and the functional specificity of Drosophila L1- and NCAM-type proteins

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Fig. 4. Overexpressions of L1- and NCAM-type proteins in the developing wing cause the same GOF phenotype. During normal wing development (A), wing margin and third vein sensilla extend axons that form two nerves, which project proximally (open arrowheads). Targeted expression of either L1-CAM, NCAM, Nrg, FasII, FasI, or a constitutively activated form of the Drosophila EGFR (k-top) (Queenan et al., 1997) induces identical alterations of axonal guidance. Axons from the wing margin sensilla often project directly towards axons of the third vein axon fascicle and fasciculate with them (open arrowheads in B, C, D, E, F, and G). Axons of the wing margin sensilla can also tangle and perforate the epithelial surface (asterisks in E and F). At early stages of sensory axon extension, numerous ectopic contacts between axon and epithelium are seen (G). These ectopic contacts are clearly reduced by a 50% gene dosage reduction of EGFR and FGFR (H). (A) MS1075/+. (B) UAS-ktop/MS1075. (C) UAS-humanNCAM 140 /MS1075. (D) Artificial GPI-anchored Nrg isoform (missing the cytoplasmic domain) in MS1096/+;UAS-NrgGPI/+. (E) UAS-mouseL1-CAM RSLE+ /MS1075. (F) UAS-humanNCAM 140 /MS1075. (G) UAS-FasI/ MS1075. (H) top/+;UAS-FasI/htl AB42 MS1075. Bar = 0.1 mm.

during the postembryonic development of the Drosophila peripheral sensory nervous system. The partially penetrant phenotypes, which have been reported for L1- and NCAM-LOF mutants in

Drosophila and different vertebrate model systems (Cohen et al., 1997; Cremer et al., 1997; Dahme et al., 1997; Fransen et al., 1997; Garcia-Alonso et al., 2000; Grenningloh et al., 1991; Hall and

Table 3 L1- and NCAM-type protein GOF phenotype is dependent on the EGFR and FGFR gene dosage

top/+

htl/+

top/+;htl/+

Nrg 180 (298C)

Nrg 167 (258C)

Nrg GPI (258C)

FasII PEST (298C)

mouseL1-CAM RLSE+ (258C)

humanNCAM 140 (258C)

FasI (258C)

ratCD2 (298C)

24.6 35/142 32.0 16/50 P = 0.35

48.4 60/124 28.7A 33/115 P = 0.002

91.4 74/81 65.3A 79/121 P = 0.0001

42.4 25/59 10.0A 2/20 P = 0.012

81.0 115/142 74.4 61/82 P = 0.31

40.6 79/195 22.8A 34/149 P = 0.0005

85.2 23/27

0.00 0/31

NT

NT

16.3 8/49 P = 0.32

21.1A 11/52 P = 0.0008

69.7A 23/33 P = 0.007

5.0A 1/22 P = 0.001

50.0A 33/66 P = 0.0001

22.3A 27/121 P = 0.0009

NT

NT

9.7A 10/103 P = 0.003

16.2A 21/130 P = 0.0001

50.0A 45/90 P = 0.0001

13.3A 2/15 P = 0.04

59.1A 68/115 P = 0.0002

7.9A 16/202 P = 0.0001

68.7 22/32 P = 0.22

NT

Penetrance (percentage of individuals displaying abnormalities) of wing sensory axon alterations. The different UAS-cDNA transgenes were expressed under the control of the GAL4 driver line MS1075 in a genetic background with either normal (100%) or half (50%) of the gene dosage for the EGFR (top/+), the FGFR (htl AB42 /+), or both FGFR and EGFR (top/+;htl AB42 /+). Animals were raised at either 258C (the combinations with Nrg 167 , Nrg GPI , FasI, mouse L1-CAM RSLE+ , and human NCAM 140 ) or 298C (the combinations with Nrg 180 , FasII PEST , and rat CD2). Higher temperatures enhance the GAL4/UAS system efficiency and, therefore, the observed phenotype. A and z represent statistically significant level of suppression or enhancement, respectively, marked by bold typeface.

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Bieber, 1997), suggest that the requirement for these neural CAMs is not absolute and that the lack of either L1- or NCAM-type proteins during nervous system development can be partially compensated for by other gene products. Moreover, considering the unique specificity of L1’s and NCAM’s homo- and heterophilic adhesive interactions, a molecular redundancy between these protein families may be unexpected. The specificities of the homophilic adhesive interactions within the L1 and the NCAM protein families have undergone considerable evolutionary changes. Drosophila Nrg and FasII exhibit a very low crossreactivity with their vertebrate homologs, L1-CAM and NCAM (Hall and Rutishauser, 1985; Hortsch et al., 1998). Although only the neuronal isoforms of human (L1-CAMRSLE+) and of Drosophila Neuroglian (Nrg180) have been directly tested for their ability to interact with each other (Hortsch et al., 1998), these results indicate that the ability of vertebrate CAMs to rescue the Nrg LOF phenotype most likely relies on homotypic adhesion, rather than on an interaction with endogenous Drosophila CAMs. This conclusion is also supported by the observation that the GOF phenotype in the wing sensory nervous system is only observed when the vertebrate transgene is expressed in both the wing epithelium and the sensory neurons. In addition, we previously showed that endogenous Nrg expression is not required for the production of the GOF axonal misguidance phenotype in the Drosophila wing (Islam et al., 2004). Although axonal growth and guidance involve a large array of different neuronal adhesion molecules, there appears to be a limited number of signaling pathways that are shared among structurally different CAM families. The two major signaling pathways, which are triggered by Ig-CAMs, involve nonreceptor tyrosine kinases or receptor tyrosine kinases, such as FGFR and EGFR (Panicker et al., 2003; Walsh and Doherty, 1997). Both of these signaling pathways may act synergistically (Niethammer et al., 2002) or in a redundant manner. Williams et al. (1994) demonstrated that L1CAM-, NCAM-, as well as N-cadherin-mediated neuronal cell adhesion all activate neuronal FGF receptors and thereby induce neurite outgrowth in vitro. This suggests that structurally different neural CAMs are capable of feeding into the same signaling pathway and that multiple adhesive specificities coordinately influence axonal growth and guidance. Previously, we demonstrated that axonal guidance in the Drosophila OSS and the wing sensory nervous system involves the Nrg-mediated activation of FGF and EGF receptors (GarciaAlonso et al., 2000; Islam et al., 2004). Constitutive activation of FGFR or EGFR can rescue the nrg 3 LOF phenotype in the OSS (Garcia-Alonso et al., 2000), and Nrg GOF axonal misguidance in the developing wing is reversed by a hypomorphic allele of the Drosophila EGF receptor (Islam et al., 2004). As we show here, the two types of neurons in the Drosophila OSS, OP and BM neurons, differ in their expression of Nrg and FasII protein and in their requirement for both proteins during axonal growth and guidance. Whereas the neuron-specific isoforms Nrg180 and FasIIPEST+ are coexpressed in OP axons, BM axons only express Nrg180, but not FasII. The surrounding epidermis, which interacts with BM but not with OP axons, expresses the nonneuronal Nrg167 isoform. The nrg LOF rescue experiments reveal strikingly different requirements for Nrg and FasII protein in the two neuronal cell populations. The requirement for Nrg in OP axons can be sustained by either the neural Nrg180 or FasIIPEST+, but not by the nonneuronal Nrg167 isoform. The two Nrg protein isoforms have identical extracellular domains and only differ in the size of their

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respective cytoplasmic domain (Hortsch et al., 1990). The capacity of FasII to fulfil the Nrg180 requirement in OP axon guidance suggests that these structurally different proteins share a redundant function in these axons. This conclusion is further supported by our observation that the partially penetrant nrg LOF OP axonal misguidance phenotype is significantly amplified by a reduction of the fasII gene dosage. Remarkably, ectopic FasIIPEST+ expression in BM neurons enhances the deleterious effect of the Nrg loss, a situation that fits within the concept of antiredundancy or opposing functional capacities (Krakauer and Plotkin, 2002). The scenario of cell-specific redundant functions of Nrg180 and FasIIPEST+ is maintained by their vertebrate homologs L1-CAM/ Nr-CAM and NCAM140, respectively. This indicates that the redundant specificities of L1 and NCAM proteins in neuronal subsets and the corresponding molecular interactions have been conserved in both CAM families over a long evolutionary time period. However, in contrast to the nonneuronal Nrg167 isoform, which exhibits an antiredundant capacity compared with Nrg180, the nonneuronal (RSLE ) vertebrate L1-CAM isoform is able to rescue the Nrg deficiency in OP axons. In contrast to Drosophila Nrg, the two vertebrate L1-CAM isoforms differ by the inclusion or exclusion of two small exons (Jouet et al., 1995; Miura et al., 1991). The insertion of the five additional amino acid residues, which are encoded by exon2, into the L1-CAM extracellular domain modifies the homo- and heterophilic functions of vertebrate L1-CAMs (De Angelis et al., 2001; Jacob et al., 2002). The inability of the human L1-CAMRSLE+ isoform to efficiently interact with Drosophila Neuroglian (Hortsch et al., 1998) suggests that the L1-CAMRSLE+ GOF phenotype is the result of homotypic molecular interactions. Moreover, the rescue of nrg 3 OP axonal phenotype by L1-CAMRSLE occurs in an Nrg deficient background, suggesting that vertebrate L1-CAMRSLE proteins are able to engage in homotypic molecular interactions in Drosophila. Interestingly, the nonneuronal human L1-CAMRSLE protein, for which De Angelis et al. (2001) postulated a lower homophilic interaction capacity, causes a much weaker GOF phenotype than the neuronal mouse L1-CAMRSLE+ isoform. Nevertheless, our results indicate that this lower homophilic binding activity of the RSLE isoform is sufficient to support the functional replacement of Nrg180 in OP axons in Drosophila. Inclusion of the cytoplasmic miniexon generates a tyrosinebased endocytosis signal (RSLEY) in the neuronal vetebrate L1CAM isoform (Kamiguchi and Lemmon, 1998). The AP-2mediated endocytosis of the neuronal L1-CAMRSLE+ isoform appears to be an important step in the activation of the MAPK signaling cascade by L1-CAM (Schaefer et al., 1999). Since neither Drosophila Nrg isoform contains an equivalent endocytosis signal in their cytoplasmic domain, Drosophila Nrg function either does not involve endocytosis or uses a different type of sorting signal than vertebrate L1 proteins. Although the analysis of the two Nrg isoforms indicates a specific requirement for Nrg180 in OSS neurons, our analysis of GOF conditions in the wing peripheral nervous system reveals an underlying common ability to activate RTK signaling. Since the Nrg-mediated activation of EGFR kinase only requires the extracellular Nrg domain for its interaction with the EGFR (Islam et al., 2004), both Nrg isoforms are able to exhibit an identical RTKdependent axonal misguidance GOF phenotype. The ability of homologous vertebrate L1- and NCAM proteins to elicit the same response in Drosophila sensory neurons indicates a common, conserved specificity to influence RTK activity and thereby to

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regulate axonal growth and guidance. However, the different ability of the neuronal versus the nonneuronal Nrg isoform to rescue the nrg LOF phenotype in the OSS indicates that Nrg-mediated axonal guidance is also regulated by cytoplasmic interactions. Since the separation of arthropods and chordates, there has been an enormous diversification in the size and organization of metazoan nervous systems. At the same time, there has also been an increase in the number of L1- and NCAM-type paralogous genes in vertebrates (but not in Drosophila), as well as structural divergence and acquisition of new specific functions within each protein family (Hinsby et al., 2004; Hortsch, 2000). Both types of proteins have conserved an average of 25–30% amino acid identity between their vertebrate and Drosophila homologues. The two groups of genes are of roughly similar size, and both have undergone independent events that resulted in the generation of different tissuespecific isoforms in Drosophila and vertebrates (Barbas et al., 1988; Grenningloh et al., 1991; Zhao and Hortsch, 1998). Although both the vertebrate and invertebrate proteins are normally coexpressed in specific axonal tracts, their respective realms of expression have shifted in insect versus vertebrate nervous systems. As a result, NCAM expression is more widespread than L1-CAM or Nr-CAM in vertebrates, while FasII is more restricted than Nrg in insects. Therefore, all these genes are evidently highly accessible to mutation and genetic drift, and the current situation most probably reflects a selective pressure to maintain NCAM- and L1-type protein coexpression in specific axonal tracts of the nervous system. Nevertheless, although both L1 and NCAM proteins have acquired many new functions in both arthropod and chordate species, it appears that they initially had at least partially overlapping roles in growth cone signaling during axon guidance. Both CAM families have apparently maintained some of these shared functions and a common specificity, including a basic function as activators of RTK signaling, over a long time period. Therefore, it seems that the functional redundancy between L1and NCAM-type proteins could constitute an important evolutionary constraint. It prevents the drift of these molecules into completely different functional entities, while at the same time, it allows their structures to further diverge and acquire separate and additional specificities. It has been proposed that functional redundancy is one mechanism for the canalization (stability after developmental perturbation and during evolution) of developmental processes (Wilkins, 1997). The requirement for a shared specificity between L1- and NCAM-type proteins in the control of RTK signaling during axon guidance might therefore reflect a requisite for redundancy that is found in any complex communication process. As originally shown by the pioneer work of C. Shannon (1948), redundancy is an essential component in any communication process for ensuring reliability by compensating the naturally occurring perturbations. Neuronal wiring is a cell communication-driven process where a highly complex set of signaling systems operates in parallel. As the number of different signals involved in axon guidance enlarged concomitant with an increase in complexity during evolution, the system noise affecting growth cone signal integration during development also increased. Unspecific adhesive interactions may also constitute a major source of noise for navigating growth cones. Therefore, cooperative redundancy might contribute to establishing a bbufferedQ physiological context required for ensuring process fidelity. We postulate that this is the reason why the ancestral functional redundancy between L1- and NCAM-type molecules has been conserved over the last 600 million years of evolution.

Experimental methods Transgene construction UAS transgenic lines for different Nrg and FasII isoforms and rat CD2 have been reported (Dunin-Borkowski and Brown, 1995; Hortsch et al., 1995; Islam et al., 2003; Lin and Goodman, 1994). The other L1-, NCAM-type, and FasI cDNAs were subcloned into the pUAST vector, and UAS-cDNA insertion lines were generated using a standard protocol for Drosophila P-element-mediated transformation. The FasI+3 cDNA was subcloned in the pUAST vector at the KpnI site. The cDNA for rat Nr-CAM was subcloned into the pUAST vector using the EcoRI site. The cDNAs for the two L1-CAM isoforms (human RSLE and mouse RSLE+) were also subcloned into the pUAST vector using the EcoRI site. Rat NCAM (140 kDa isoform VASE ) was shuttled through the pBluescript II KS + plasmid to obtain the Xho–XbaI sites used for ligation into the pUAST vector. Human NCAM (140 kDa, VASE ) was first subcloned into pBluescript II KS + and subsequently transferred into pUAST using the XhoI–XbaI restriction sites. All pUAST clones containing vertebrate cDNAs were sequenced 5V to ensure correct direction of insert and presence of ATG. The distance from the pUAST polylinker (EcoRI site) to the translation start codon in individual cDNAs varies from 20 to 546 bp. The different UAS insertions for the vertebrate L1- and NCAM-type molecules were confirmed to drive protein expression in combination with the GAL4 insertion line MS1075 using specific commercially available antibodies (Santa Cruz Biotechnology Inc.). Genetic crosses and phenotype analysis Mutants and GAL4 driver insertions (MS1096 and MS1075) have already been reported (Garcia-Alonso et al., 2000; Grenningloh et al., 1991; Lindsley and Zimm, 1992). The A101 insertion line carries a P[lacZ] insertion at the neuralized locus (Usui and Kimura, 1993). Transgenic UAS-L1-type and UAS-NCAM-type strains were maintained balanced with the TM6B, CyO, or T(2;3)SM6a-TM6B balancer chromosomes. The TM6B balancer chromosome allows identification of the pupae harboring the transgene for those insertions in the third chromosome, while the T(2;3)SM6a-TM6B balancer chromosome allows the identification of pupae with insertions in the second chromosome. Crosses between the GAL4 driver line and the UAS-cDNA lines were maintained at 258C or 298C as explained in the Results. Temperature shifts and phenotypic analysis in rescue crosses involving nrg 3 were performed as previously described (Garcia-Alonso et al., 2000). nrg 3 rescue crosses were established between nrg 3 ;MS1075 virgin females and w;UAS-cDNA males. Crawling male larvae ready for puparium formation, previously shifted at 298C (GarciaAlonso et al., 2000), were isolated from the cross and maintained at 298C for further 30 to 40 h of pupal development, and then fixed for immunohistochemistry. As parallel controls, we used both nrg 3 ;MS1075/+ as well as nrg 3 ;UAS-cDNA/+ individuals (data not shown) for each one of the different transgenic lines (no modification of the nrg 3 phenotype was observed in any case). Immunostainings of pupal heads and wings with MAbs 22C10, 1D4, and BP104 were performed as previously described (GarciaAlonso et al., 2000). Statistical significance of penetrance values was calculated using the two-tailed Fisher exact test for comparison of categorical data.

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Acknowledgments We thank S. Kenwrick, C. Faivre-Sarrailh, and M. Schachner, for providing vertebrate cDNAs, and J. Urban for the UAS-CD2 insertion line. We also thank N. Crisp for technical assistance and A. Nieto, J. Bagun˜a, I. Farin˜as, and A. Garcia-Bellido for a critical reading of the manuscript. This work was supported by an EMBO short-term fellowship to L.V.K.; an FPI predoctoral fellowship to E.V.; grants from Vera and Carl Johan Michelsen’s Foundation and the Danish Cancer Society to E.B.; a grant from the Lundbeck Foundation to V.B.; NICHD HD29388, NSF IBN-0132819, and SCRF No.1797 grants to M.H.; and by the MCYT SAF2001-1628 (part from FEDER) grant to L.G-A.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mcn.2004.09.003.

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