Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering

Article

Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering Graphical Abstract

Authors Lina Liu, Yao Tian, Xiao-yan Zhang, Xinwang Zhang, Tao Li, Wei Xie, Junhai Han

Correspondence [email protected]

In Brief Columnar restriction of neurites is commonly observed in the nervous system. Liu, Tian et al. demonstrate that a-neurexin promotes ephrin clustering via its intercellular domain and that ephrin/ Eph signaling from adjacent L4 neurons restricts L4 axon branches into columns.

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DNrx is enriched in L4 axon terminals and restricts axonal branching in columns

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Defective columnar restriction in dnrx mutants is due to impaired ephrin clustering

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DNrx associates with ephrin and promotes ephrin clustering

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DNlg4 initiates DNrx clustering and subsequently induces ephrin clustering

Liu et al., 2017, Developmental Cell 41, 1–13 April 10, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2017.03.004

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

Developmental Cell

Article

Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering Lina Liu,1,3 Yao Tian,1,3 Xiao-yan Zhang,1 Xinwang Zhang,1 Tao Li,1 Wei Xie,1,2 and Junhai Han1,2,4,* 1Institute of Life Sciences, Key Laboratory of Developmental Genes and Human Disease, Southeast University, 2 Sipailou Road, Nanjing 210096, China 2Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China 3Co-first author 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.03.004

SUMMARY

Columnar restriction of neurites is critical for forming nonoverlapping receptive fields and preserving spatial sensory information from the periphery in both vertebrate and invertebrate nervous systems, but the underlying molecular mechanisms remain largely unknown. Here, we demonstrate that Drosophila homolog of a-neurexin (DNrx) plays an essential role in columnar restriction during L4 axon branching. Depletion of DNrx from L4 neurons resulted in misprojection of L4 axonal branches into neighboring columns due to impaired ephrin clustering. The proper ephrin clustering requires its interaction with the intracellular region of DNrx. Furthermore, we find that Drosophila neuroligin 4 (DNlg4) in Tm2 neurons binds to DNrx and initiates DNrx clustering in L4 neurons, which subsequently induces ephrin clustering. Our study demonstrates that DNrx promotes ephrin clustering and reveals that ephrin/Eph signaling from adjacent L4 neurons restricts axonal branches of L4 neurons in columns.

INTRODUCTION The organization of neuronal arbors into layers and columns is a common feature of both vertebrate and invertebrate nervous systems (Mountcastle, 1997). Columnar restriction of sensory neurons preserves the high-fidelity transmission of spatial information from the periphery to more central regions of the brain (Clandinin and Zipursky, 2002), which is mediated by contactdependent repulsion (Grueber et al., 2003; Perry and Linden, 1982; Wassle et al., 1981) and intrinsic regulation of neurite growth (Lee et al., 2001; Ting et al., 2007). However, the molecular mechanisms of this process are not fully understood. The Drosophila visual system provides an excellent model for exploring the genetic and molecular bases for precise columnar arrangement (Kunes and Steller, 1993). The compound eye of the fly comprises approximately 750 ommatidia, each containing eight photoreceptor neurons (R cells, R1–R8), which project to the brain and make connections within two neuropils, the lamina

and the medulla. The medulla is arranged into
750 columns, each of which receives projections from one R7, one R8, and five lamina (L1–L5) neurons, and the medulla is also divided into ten parallel layers (M1–M10) (Fischbach and Dittrich, 1989). This topographic mapping is guided by long- or short-range cues between axons and the target layer or between different axons (Ting et al., 2005). Although some molecular studies have made progress in understanding how axons select their target layers (Clandinin and Zipursky, 2002; Petrovic and Hummel, 2008), much less is known about the mechanism of columnar restriction. The activin receptor Baboon and the nuclear import adaptor importin-a3 have been identified to restrict R7 terminal processes to a single column (Ting et al., 2007). Dscam2 was identified as a membrane protein for L1 columnar restriction in Drosophila (Millard et al., 2007). In addition, N-cadherin has been shown to restrict L5 terminal processes to a single column (Nern et al., 2008). However, the molecules mediating the columnar restriction of other lamina neurons remain unknown. Ephrin guidance factors and their receptors (Ephs) act as repellents regulating axon guidance (Egea and Klein, 2007). Eph/ ephrin signaling enables bidirectional cell-to-cell communication, whereby ephrins act as classical ligands for Ephs to initiate forward signaling and also act as receptors for Ephs for reverse signaling (Egea and Klein, 2007; Klein, 2009). They are divided into two main subfamilies of ligand/receptor couples, ephrin-A/ EphA and ephrin-B/EphB, on the basis of their specific structures and binding affinities (Flanagan and Vanderhaeghen, 1998). Ephrin-B1 has been shown to control the columnar distribution of cortical pyramidal neurons by restricting their tangential migration (Dimidschstein et al., 2013). In the Drosophila visual system, the B-class ephrin ortholog is required for topographic map formation (Bossing and Brand, 2002; Dearborn et al., 2002). However, whether ephrin/Eph signaling is involved in the columnar restriction of lamina neurons is unknown. Neurexins function in the formation and transmission of synapses (Dean et al., 2003; Li et al., 2015; Missler et al., 2003; Qiu et al., 2007; Scheiffele, 2003; Zeng et al., 2007) and were also shown to interact directly with neuroligin on the postsynaptic membrane to influence dendrite growth and stability in Xenopus (Chen et al., 2010). In addition, a-neurexin knockout mice exhibit shortened distal dendritic branches (Dudanova et al., 2007). These results suggest that neurexins also play a role in early neural development. Here, we show that Drosophila neurexin (DNrx) is enriched in the axon terminals and the interstitial processes of Developmental Cell 41, 1–13, April 10, 2017 ª 2017 Elsevier Inc. 1

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Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

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Figure 1. DNrx Is Enriched in the Axon Terminals and Interstitial Processes of Developing Lamina Neurons (A) DNrx distribution in the developing outer medulla. Pupae and adult heads were stained with anti-DNrx (green) and 24B10 (red, for R cell axons) antibodies. Single confocal sections of DNrx staining in the wild-type at indicated times after puparium formation (APF) are shown. Positions of R8 and R7 growth cones are indicated by dashed lines. Scale bars, 10 mm. Graphs showing layer distribution of the anti-DNrx staining intensity averaged over six adjacent columns are presented as mean ± SEM on the right. The layer positions of the R8 and R7 growth cones are marked by dashed lines and the top of medulla are indicated by triangles in the graphs. (B) DNrx expression in developing lamina neurons. Pupae heads were collected at 48 hr APF and stained with anti-DNrx (red) and anti-GFP (green, for lamina axons) antibodies. Single confocal sections show DNrx staining in control (red, left) and Ln > dnrxRNAi (red, right) flies, respectively. Note that non-Ln-projection regions are normal and can be used as an internal control. Scale bars, 10 mm. (C) DNrx expression in developing L4 neurons. Pupae heads were collected at 80 hr APF and stained with anti-DNrx (red) and anti-GFP (green, for L4 axons) antibodies. Single confocal sections show DNrx staining in control (red, left) and L4>dnrxRNAi (red, right) flies, respectively. Positions of the axon terminals and interstitial processes of L4 neurons are indicated. Note that non-L4-projection regions are normal and can be used as an internal control. Scale bars, 10 mm. (D) Immunostaining images indicating that full-length DNrx distributes into the axon terminals and interstitial processes of L4 neurons. Adult heads were stained with anti-DNrx (red) and anti-GFP (green, for L4 axons) antibodies. Scale bars, 5 mm. See also Figure S1.

L4 lamina neurons, and the loss of DNrx results in the extension of L4 axon branches into neighboring columns. We further identify that DNrx associates with and promotes clustering of the guidance factor ephrin, which restricts axonal branching within a single column. RESULTS DNrx Is Enriched in Axon Terminals and Interstitial Processes of Developing Lamina Neurons Neurexins are cell-adhesion molecules involved in synapse formation (Dean et al., 2003; Qiu et al., 2007; Scheiffele, 2003; Zeng et al., 2007) and synaptic transmission (Missler et al., 2003). Although multiple a-neurexin genes exist in vertebrate genomes, only a single a-neurexin gene (CG7050, dnrx, gene ID: 42646) has been identified in the Drosophila genome (Li et al., 2007; Zeng et al., 2007). We generated anti-DNrx antibodies and validated them by western blotting (Figure S1A). Antibody staining revealed that DNrx was expressed in the medulla at 2 Developmental Cell 41, 1–13, April 10, 2017

24 hr after puparium formation (APF) and was enriched between R7 and R8 growth cones with a parallel column arrangement (Figures 1A and S1B). At 48 hr APF, the DNrx-rich region was divided into two layers; one fell behind the R7 growth-cone layer and the other was ahead of the R8 growth-cone layer (Figures 1A and S1B). At 55 hr APF, the DNrx-rich region was further divided into four layers separated by three zones of weaker DNrx staining (Figures 1A and S1B). In adults, DNrx localized in layers M1–M4 but was absent or weakly existed in layers M5 and M6 (Figures 1A and S1B). These data suggest there is dynamic expression of DNrx in the developmental processes of different cell types, in specific subcellular regions, or at specific cell contacts. Next, we sought to identify the cellular origins of the DNrx protein. Lamina neuron axons follow R7 axons and extend into the medulla, and terminate between the temporary R8 and R7 layers. Therefore, we wondered whether DNrx was expressed in lamina neurons. We labeled lamina neurons with membranetethered GFP (Ln-GAL4, UAS::mCD8-GFP) and found that DNrx staining overlapped with lamina neuron projections

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

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Figure 2. DNrx Restricts L4 Axon Branches to a Single Column (A) Confocal images (left) and schematics (middle) showing medulla terminals of wild-type (WT) (upper panels) and dnrxD83/273 mutant L4 neurons (lower panels). Confocal images showing L4 terminals in cross-sections of the M4 layer are presented on the right. Adult heads were stained with anti-GFP (green, for L4 axons) and 24B10 (red, for R cell axons) antibodies. WT L4 axons arborized in the M2 and M4 layers of the medulla and were restricted to a single column. The mutant L4 axons that projected into neighboring columns are indicated by arrows. Scale bars, 5 mm. (B) Percentages of L4 axons extending into neighboring columns in each genotype. Data are presented as mean ± SEM. WT, n = 22; dnrx273/+, n = 10; dnrxD83, n = 12; dnrx273, n = 8; dnrxD83/273, n = 28. ***p < 0.001; ns, not significant. (C) Confocal images showing the medulla terminals of single WT and dnrx273 mutant L4 neurons (upper panels) and their terminals in cross-sections of the M4 layer (lower panels). L4 MARCM clones were generated with dac-FLP and stained with anti-GFP (green) and 24B10 (red, for R cell axons) antibodies. Scale bars, 5 mm. (D and E) Percentages of L4 axon branches extending into neighboring columns in M2 and M4 layers in the WT and dnrx273 mutants (D), and percentages of L4 neurons showing abnormal bifurcation in WT and dnrx273 mutants (E). Data are presented as mean ± SEM. The total number of examined WT L4 clones is 150, while the total number of examined mutant L4 clones is 145. ***p < 0.001. See also Figure S2.

(Figure 1B). Moreover, knockdown of dnrx with Ln-GAL4 significantly reduced DNrx staining in lamina neuron projections but not in areas without lamina neuron projections (Figure 1B). We further labeled individual subsets of lamina neurons with mCD8-GFP and examined the expression of DNrx in the outer medulla at 80 hr APF, the time at which five subsets of lamina neurons have terminated into diverse layers and are easily distinguishable. We found that DNrx expression overlapped with lamina projections in layers M1–M4, but its expression was weak or absent in layer M5 at later pupal stages (Figure S1C). To provide further evidence showing that DNrx is expressed in L4 neurons, we labeled them with mCD8-GFP and stained the heads of laterstage pupae with anti-DNrx antibodies. We found that DNrx staining was condensed in the axon terminals and interstitial branches of L4 neurons, whereas the knockdown of dnrx with L4-GAL4 reduced DNrx staining specifically in L4 neuron projections (Figure 1C). To further explore the subcellular distribution of DNrx in L4 neurons, we generated a full-length neurexin transgene

(p[UAS-DNrxFL]) and expressed DNrxFL in L4 neurons in a dnrx mutant background. Interestingly, we found that DNrxFL accumulated in the axon terminals and interstitial branches of L4 neurons (Figure 1D). Taken together, these data demonstrate that the DNrx is expressed in L4 neurons and is enriched in the axon terminals and interstitial branches of lamina neurons, strongly suggesting that it plays a role in axonal targeting. DNrx Restricts L4 Axon Branches to a Single Column To assess the role of DNrx in axonal targeting, we labeled individual subsets of lamina neurons using subset-specific GAL4 drivers. We found that the axons of all lamina neurons and R7/R8 cells projected to appropriate layers in dnrxD83/dnrx273 mutants (Figures 2A and S2A), indicating that DNrx is not required for the targeting of all lamina neurons and R7/R8 cells to specific layers. Interestingly, we found that L4 axons of dnrxD83/dnrx273 mutants were no longer restricted within a single column and often extended into neighboring columns, although these axons still terminated in M4 layers (Figures 2A and 2B). Furthermore, the defective Developmental Cell 41, 1–13, April 10, 2017 3

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

columnar restriction was observed in dnrxD83 and dnrx273 single mutant flies, but not in dnrx273 heterozygotes (Figure 2B). To further assess the phenotypes of L4 neurons in the mutants, we labeled single L4 neurons using the MARCM technique (Lee and Luo, 1999). To induce selective recombination in lamina precursor cells, we expressed FLP recombinase under the control of the Dachshund (Dac) enhancer (Millard et al., 2007). Single-cell labeling revealed that wild-type L4 axons terminated and bifurcated in the M4 layer but their interstitial branches terminated in the M2 layer (Figure 2C). Moreover, terminal and interstitial branches of wild-type L4 neurons were restricted to a signal column (Figure 2C). Although the terminal and interstitial branches were in the correct layers (M4 and M2, respectively), 17.9% ± 2.0% of terminal branches and 19.3% ± 2.8% of interstitial branches of dnrx273 mutant L4 neurons were no longer restricted to a single column and often extended into neighboring columns (Figures 2C and 2D). In addition, compared with wild-type L4 neurons, dnrx273 mutant axons frequently bifurcated in the M2 or M3 layer, with their terminal branches invading adjacent columns (8% ± 1.3% versus 18.6% ± 2.8%, Figure 2E). In contrast, single-cell labeling revealed that dnrx mutant L5 axons exhibited morphologies comparable with those of wildtype L5 axons (Figure S2B). These observations indicate that DNrx is required for L4 columnar restriction. Given that L4 neurons in the single mutant were generated in a wild-type background, these results also indicate that DNrx restricts axon branching within columns in a cell-autonomous manner. Defective Columnar Restriction in dnrx Mutant Occurs at Mid-pupal Stage To elucidate the mechanisms by which DNrx restricts L4 branching, we first determined the developmental requirement for DNrx by comparing axonal branching and targeting in wild-type and dnrx mutant L4 neurons. We applied Ap-Gal4 with MARCM, as it efficiently labels L4 neurons throughout development (Nern et al., 2008). Consistent with the results from a previous report (Nern et al., 2008), wild-type L4 axons formed complex interstitial branches in the M2 layer and terminal branches in the M4 layer at 48 hr APF, and these branches spread evenly into the column over the ensuing 12 hr (Figure 3A). Between 60 and 80 hr APF, these branches retracted gradually, and only two or three short terminal branches and a few interstitial branches remained at 80 hr APF (Figure 3A). The morphologies of mutant L4 axons were comparable with those of wild-type L4 neurons at 48 hr APF, in which the interstitial branches resided in the M2 layer and the terminal branches localized to the M4 layer (Figure 3B). At 55 hr APF, the mutant interstitial and terminal branches extended in one direction, whereas branches in other directions retracted (Figure 3B). As the pupae developed, the extended branches in the mutant L4 axons further elongated and the branches in other directions further retracted (Figure 3B). At a later pupal stage (72 and 80 hr APF), the mutant interstitial and terminal branches frequently invaded adjacent columns (Figure 3B). We also applied RNAi against DNrx using R31C06-GAL4, which drives expression of GAL4 in L4 neurons later than 40 hr APF (Figure S3). Interestingly, the depletion of DNrx with R31C06-GAL4 resulted in the projection of L4 axons into adjacent columns (Figure 3C). This defective columnar restriction was completely restored by expressing DNrxFL with R31C06-Gal4 in 4 Developmental Cell 41, 1–13, April 10, 2017

dnrx mutants (Figure 3D). In contrast, expressing DNrxFL with c739-GAL4, which drives expression in multiple brain regions, failed to rescue the defective columnar restriction in dnrx mutants (Figure S4). To further narrow the time window in which DNrx regulates columnar restriction, we generated UAS-mCD8::GFP/ tubP-GAL80ts;R31C06-GAL4,dnrxD83/UAS-DNrxFL,dnrxD83 flies in which UAS::DNrx expression is induced at 30 C by the ubiquitously expressed temperature-sensitive GAL80ts (McGuire et al., 2004). Interestingly, defective columnar restriction was largely restored in the flies exposed to 30 C from 48 hr to 60 hr APF but not from 80 hr to 92 hr APF (Figure 3E). Taken together, these results demonstrate that DNrx regulates columnar restriction at the mid-pupal stage. DNrx Associates with Ephrin In Vivo It has been proposed that columnar restriction is regulated by the intrinsic control of neurite growth and by local repulsions between axon processes, which are regulated in part by ephrin/Eph signaling. In our previous yeast two-hybrid screening using the intracellular region of DNrx as a bait, we isolated a cDNA encoding the intracellular region of a Drosophila B-class ephrin ortholog (605–652 amino acids, accession number NP_726583.1, Figure 4A) (Li et al., 2015). To investigate the potential interaction between DNrx and ephrin, we generated antibodies that recognize the intracellular domain of Drosophila ephrin. Western blotting showed three major bands (72 [predicted size of Drosophila ephrin], 69, and 91 kDa) in isolated wild-type pupal eyes that were significantly reduced in isolated Ln-GAL4/UAS-ephrinRNAi fly eyes (Figure 4B). Given that only one ephrin transcript was identified in Drosophila (Bossing and Brand, 2002), the 91- and 69-kDa bands might represent post-translational modifications and protein cleavage, respectively. Two additional bands (54 and 52 kDa) were expressed at comparable levels in these two fly eyes (Figure 4B), suggesting that they might be nonspecific bands. Immunostaining experiments revealed that the ephrinrich region was divided into three (upper, middle, and lower) layers separated by zones of weaker ephrin staining at 55 hr APF (Figure 4C). Interestingly, compared with wild-type flies, ephrin staining was significantly reduced in all three layers in Ln-GAL4/UAS-ephrinRNAi flies, whereas staining was reduced only in the upper and middle layers in L4-GAL4/UAS-ephrinRNAi flies (Figures 4C and 4D). These results demonstrate that ephrin is also expressed in L4 neurons. To provide evidence showing that DNrx interacts with ephrin in vivo, we performed immunostaining and found that Drosophila ephrin co-localized with DNrx in the outer medulla at 48 hr APF (Figure 4E). Co-immunoprecipitation experiments demonstrated an in vivo interaction between DNrx and the 72-kDa ephrin (Figure 4F). These results suggest that DNrx physically associates with ephrin in vivo. The intracellular region of DNrx contains 122 amino acids, including a functional PDZ-binding motif (Figure 4G). To determine which amino acids within the intracellular region of DNrx interact with ephrin, we performed pull-down assays using several constructs encoding truncated DNrx fragments fused to glutathione S-transferase (GST) (Figures 4G and S5). The results revealed that a purified ephrin C terminus (ephrinC, amino acids 454–652) bound with the amino acids fragments 1,732–1,787, 1,732–1,813, 1,732–1,833, and 1,732–1,837

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

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of DNrx, but not with the 1,732–1,759 fragment (Figures 4G and S5). In addition, binding to the 1,732–1,787 amino acid fragment was reduced in comparison with the 1,732–1,837 fragment. To further validate the ephrin-binding sites in DNrx, we expressed and purified two mutant fragments (DNrxD17601787 and DNrxD17601813) with amino acids 1,760–1,787 and 1,760– 1,813 of DNrx deleted, respectively. Although DNrxD17601787 showed a weak ability to bind with ephrin, DNrxD17601813 failed to bind with ephrin (Figures 4G and S5). These observations indicate that amino acids 1,760–1,813 in the intracellular region of DNrx mediate the interaction with ephrin. Abnormal Columnar Restriction in dnrx Mutant Is Due to Impaired Ephrin Function On the basis of the findings described above, we tested whether DNrx restricted axonal branching in columns by modulating ephrin function. Thus, we next compared ephrin distributions in

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Figure 3. Defective Columnar Restriction in dnrx Mutant Occurs at the Mid-pupal Stage (A and B) Medulla terminals of single wild-type (WT) (A) and dnrx273 mutant (B) L4 neurons at indicated times APF. Column boundaries are determined based on 24B10 staining. Scale bars, 5 mm. (C) Confocal images showing medulla terminals of L4 neurons in control (upper panel) and L4>dnrxRNAi (lower panel) flies. Adult heads are stained with anti-GFP (green) and 24B10 (red, for R cell axons) antibodies. The L4 axons that project into neighboring columns are indicated by arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. Control, n = 11; L4>dnrxRNAi, n = 13. ***p < 0.001. (D) Confocal images showing medulla terminals of L4 neurons in dnrx mutant (upper panel) and rescue (lower panel) flies. Adult heads are stained with anti-GFP (green) and 24B10 (red, for R cell axons) antibodies. The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. dnrx, n = 28; rescue, n = 18. ***p < 0.001. (E) Confocal images showing L4 projections in the flies expressing the UAS-DNrx and temperature-sensitive GAL80ts in L4 neurons. Entrainment temperatures and entrainment protocol are shown at the bottom. 120 hr APF represents the time point of eclosion. After eclosion, all flies were entrained at 30 C for 24 hr to permit GFP expression. The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each entrainment are presented on the right. Data are presented as mean ± SEM. no hs, n = 7; 48–60, n = 8; 80–92, n = 8. ***p < 0.001; ns, not significant. See also Figures S3 and S4.

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wild-type and dnrx mutant L4 axons. In wild-type flies, ephrin was weakly detected in the growth cones of lamina neurons at 24 hr APF (Figure 5A). As the pupae developed (48 and 55 hr APF), ephrin protein levels gradually increased in the outer medulla and condensed in axon terminals and interstitial processes (Figures 5A and 5B). Ephrin levels in the outer medulla fell and were sustained at a low level after 72 hr APF (Figures 5A and 5B). These results reveal that ephrin is expressed in a distinct stage-specific fashion in the developing medulla neuropil, and indicate that the dynamic expression of ephrin in the outer medulla correlates with L4 axon branching. In contrast to wildtype flies, dnrx mutants showed a diffuse ephrin distribution throughout development and reduced ephrin levels in axon terminals and interstitial processes at the mid-pupal stage (48 and 55 hr APF) (Figures 5A and 5B). We excluded the possibility that reduced ephrin levels in axon terminals and interstitial processes at the mid-pupal stage were due to decreased mRNA Developmental Cell 41, 1–13, April 10, 2017 5

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Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

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Figure 4. DNrx Associates with Ephrin In Vivo (A) Yeast two-hybrid screening identified one cDNA for ephrin, E10, interacting with neurexin. The encoded region of E10 is shown at the top. V1, pGBKT7; V2, pGADT7-RecAB. (B) Ephrin expression levels in isolated wild-type and Ln-GAL4/UAS-ephrinRNAi medullas. Each lane was loaded with isolated medullas from five flies. The ephrin bands are indicated with arrows, and two nonspecific bands are indicted with an open arrow. (C) The expression and distribution of ephrin in the outer medulla in wild-type and RNAi flies. Pupae heads were collected at 55 hr APF and double-stained with anti-ephrin (green) and 24B10 (red, for R cell axons) antibodies. Three ephrin staining layers are marked: U, upper layer; M, middle layer; L, lower layer. Scale bars, 5 mm. (D) Quantification of ephrin levels in each layer in each genotype. Five flies of each genotype were examined, and more than four sections were checked in each medulla. Data are presented as mean ± SEM. ***p < 0.001; ns, not significant. (E) Endogenous ephrin co-localizes with DNrx in the outer medulla. Wild-type pupae heads were collected at 48 hr APF and stained with anti-DNrx (red) and antiephrin antibody (green) antibodies. Scale bar, 10 mm. (F) Co-immunoprecipitation of DNrx and ephrin in vivo. Pupae head extracts were immunoprecipitated with anti-DNrx antibodies. The precipitates, as well as a portion (1% of the input for anti-DNrx IP) of pupae head extracts, were subjected to western blotting with anti-ephrin and anti-PLC (for the negative control) antibodies. Note that immunoglobulin G (IgG) can be used as an internal loading control. WT, wild-type. (G) A pull-down assay mapped residues 1,760–1,813 as an ephrin-binding site of DNrx. The pull-down samples and 2% of purified MBP-ephrinC were loaded for western blotting. Various GST-DNrx fusion fragments used for the pull-down assay were stained with Coomassie blue and indicated with red stars. Other low (legend continued on next page)

6 Developmental Cell 41, 1–13, April 10, 2017

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transcription, because dnrx mutants had normal levels of ephrin mRNAs throughout pupal development (Figure 5C). To provide further evidence that reduced ephrin levels result from a loss of DNrx, we generated dnrx mutant clones using the MARCM technique, which enables the generation and visualization of individual homozygous dnrx mutant lamina neurons within heterozygous tissue (Lee and Luo, 1999). Interestingly, compared with neighboring heterozygous clones, dnrx mutant clones showed reduced ephrin staining in upper and middle layers, but not in the lower layer (Figure 5D). Given that the activity of ephrin relies on its clustering (Davis et al., 1994), we suspected that the diffuse distribution and reduced expression of ephrin at the mid-pupal stage were responsible for the defective L4 columnar restriction in dnrx mutants. To test this hypothesis, we depleted ephrin from L4 neurons and examined their projections. Interestingly, the frequency of L4 axon branching into neighboring columns was higher with knockdown of ephrin using the R31C06-GAL4 driver than in dnrx mutant flies (Figure 5E). We also performed RNAi against ephrin with the R31C06-GAL4 driver in dnrx mutant flies. Interestingly, R31C06-GAL4;UAS-ephrinRNAi;dnrx flies showed a frequency of L4 axonal branching into neighboring columns that was comparable with that of R31C06-GAL4/UAS-ephrinRNAi flies (Figure 5E). Taken together, these data indicate that the defective L4 columnar restriction in dnrx mutants is due to impaired ephrin function at the mid-pupal stage. A previous study showed that the ephrin receptor, Eph (gene ID: 43803), is concentrated in the synaptic neuropil of the outer medulla at 45 hr APF (Dearborn et al., 2002). We next attempted to anatomically map Eph-dependent L4 columnar restriction. We labeled L4 neurons with tdTomato using a lexA/lexAop system and RNAi against Eph using the Gal4/UAS system. Seven Gal4 drivers that label different types of cells in the outer medulla were tested. We found that Ln-GAL4 and R31C06-GAL4 lines recaptured the defective columnar restriction that was observed in dnrx mutant flies (Figure 5F). Both lines that label L4 neurons, cha-GAL4 and ort-GAL4 lines (Figure S6) (Takemura et al., 2011; Ting et al., 2011, 2014), recaptured the defective columnar restriction that was observed in dnrx mutant flies (Figure 5F). By contrast, GAL4 lines that label other types of cells in the outer medulla (e.g., repo-GAL4, R7-GAL4, and otd-GAL4) showed normal columnar restriction (Figure 5F). Interestingly, knockdown of Eph with Ln-GAL4 resulted in the extension of L4 axons into the M5 layer (open arrows, Figure 5F), suggesting that ephrin/Eph signaling is also required for the layer targeting. Taken together, these data indicate that the expression of Eph in adjacent L4 neurons restricts L4 axons to a column. DNrx/Ephrin Interaction Promotes Ephrin Clustering A previous study showed that the interaction between the PDZ-binding motif of ephrin-B and the PDZ-containing protein PICK1 induces the clustering of ephrin-B in vivo (Torres et al., 1998). However, the cytoplasmic tail of Drosophila ephrin does not contain a PDZ-binding consensus sequence (Bossing and

Brand, 2002), suggesting that an alternative mechanism exists for Drosophila ephrin clustering. Thus, we suspected that a DNrx/ephrin interaction might promote ephrin clustering in Drosophila. As cell transfection has been successfully utilized in the past to demonstrate that ephrin-B forms co-clusters (Torres et al., 1998), we transfected S2R+ cells with full-length ephrin and observed a diffuse staining pattern (Figure 6A). We first confirmed that both expressed DNrxFL and expressed DNrxD17601813 were able to localize on the membrane (Figure S7). Interestingly, we observed a dramatic co-clustering when ephrin was co-expressed with full-length DNrx (DNrxFL, Figure 6A). By contrast, co-clustering was significantly reduced when ephrin was co-expressed with the truncated DNrxD17601813, which does not contain the ephrin-binding site (Figure 6A). To provide further evidence that this interaction promotes ephrin clustering and subsequent L4 columnar restriction, we generated a p[UAS::DNrxD17601813] transgene, in which the ephrin-binding site of DNrx was deleted. The expression of full-length DNrx, but not the truncated DNrxD17601813, in dnrx mutant L4 neurons largely restored ephrin staining in the upper and middle layers at 55 hr APF (Figure 6B). Moreover, columnar restriction was restored in dnrx mutants by R31C06-GAL4driven expression of full-length DNrx but not the truncated DNrxD17601813 (Figure 6C). Taken together, these data demonstrate that the DNrx/ephrin interaction is critical for ephrin clustering and subsequent columnar restriction. DNlg4 Facilitates DNrx Clustering and Regulates L4 Columnar Restriction Next, we asked how DNrx promotes ephrin clustering. Postsynaptic neuroligin multimers have been shown to initially cluster axonal Neurexin, which in turn nucleates the assembly of a cytoplasmic scaffold (Dean et al., 2003). Thus, we wondered whether a neurexin/neuroligin interaction promotes neurexin clustering and subsequent ephrin clustering. To test this hypothesis, we generated transgenic flies expressing truncated DNrx, p[UASDNrxDN], in which the extracellular region of DNrx was deleted. We found that expressing DNrxDN in L4 neurons failed to restore L4 columnar restriction in dnrx mutant flies (Figure 7A). These data indicate that the extracellular domain of DNrx is required for restricting L4 axon branches to a column. Four Drosophila neuroligin homologs, dnlg1 (CG31146, gene ID: 40913), dnlg2 (CG13772, gene ID: 33962), dnlg3 (CG34127, gene ID: 40912), and dnlg4 (CG34139, gene ID: 42402), have been identified (Banovic et al., 2010; Knight et al., 2011; Li et al., 2013; Sun et al., 2011; Xing et al., 2014). To identify which neuroligin is required for axonal neurexin clustering and restricting L4 axon branches to columns, we examined the projections of L4 neurons in these mutant flies. Although both dnlg2 and dnlg3 mutants showed normal column restriction for L4 axon branches (Figure S8A), L4 axonal branches of dnlg4 mutants frequently invaded neighboring columns (Figure 7B), phenocopying the defective columnar restriction observed in dnrx mutant flies. Antibody staining confirmed that endogenous expression

molecular weight bands in each lane are degraded products. Upper panel: the encoded regions of DNrx fragments; middle panel: Coomassie blue staining of various GST-DNrx fragments and western blots of pull-down assays; lower panel: quantification of ephrin-binding efficiencies for each GST-DNrx fragment. Three sets of independent data were averaged, and the data are presented as mean ± SEM. See also Figure S5.

Developmental Cell 41, 1–13, April 10, 2017 7

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Figure 5. Abnormal Columnar Restriction in dnrx Mutant Is Due to Impaired Ephrin Function

(A) The expression and distribution of ephrin in the outer medulla in wild-type (WT) and dnrxD83/273 mutants at indicated times APF. Pupae heads were doublestained with anti-ephrin (green) and 24B10 (red, for R cell axons) antibodies. Scale bars, 10 mm. (B) Relative ephrin protein levels in the outer medulla at indicated times APF. Eight flies of each genotype were examined, and more than six sections were checked in each medulla. Data are presented as mean ± SEM. ***p < 0.001; ns, not significant. (legend continued on next page)

8 Developmental Cell 41, 1–13, April 10, 2017

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of DNlg4 in the outer medulla at 60 hr APF (Figure 7C). These data support the role of DNlg4 in restricting L4 axonal branching. However, the depletion of DNlg4 from individual lamina neurons resulted in normal axonal projections of these lamina neurons (Figure S8B), suggesting that DNlg4 is not required for the layer targeting of these lamina neurons. Next, we tried to identify the postsynaptic source of DNlg4 that mediates presynaptic DNrx clustering in L4 neurons. As DNlg4 is broadly expressed in the outer medulla, several GAL4 lines encompassing DNlg4 expression patterns were selected for RNAi knockdown experiments. We labeled L4 neurons with tdTomato using the lexA/lexAop system and expressed RNAi against dnlg4 using the Gal4/UAS system. Interestingly, expression with the cha-GAL4 and ort-GAL4 lines, which label Tm2 neurons (Takemura et al., 2011; Ting et al., 2011, 2014), recapitulated the defective columnar restriction observed in dnrx mutant flies (Figure S8C). By contrast, the R31C06-GAL4 line showed normal columnar restriction (Figure S8C). Moreover, the knockdown of dnlg4 with the Tm2 neuron-specific otd-GAL4 resulted in defective columnar restriction (Figure 7B). Conversely, columnar restriction was largely restored by otd-GAL4 in dnlg4 mutant flies (Figure 7B). These results are consistent with a previous report which revealed that Tm2’s dendrites form apparent contacts with the L4 axons (Takemura et al., 2011). Taken together, these data indicate that Tm2 neuron-expressed DNlg4 mediates presynaptic DNrx clustering in L4 neurons. To further investigate whether DNlg4 restricts L4 axon branches within columns through the clustering of DNrx and ephrin, we directly examined the distributions of DNrx and ephrin in dnlg4 mutant flies. Immunostaining revealed that DNrx and ephrin showed diffuse distributions and reduced protein levels in the outer medulla of dnlg4 mutants at the mid-pupal stage (Figures 7D and 7E). We also generated dnlg4,dnrx double-mutant flies and assessed the axon projections of their L4 neurons. Compared with dnrx mutant flies, dnlg4,dnrx double mutants showed normal frequencies of L4 axonal branching into neighboring columns (Figure 7F). Moreover, depletion of ephrin in dnlg4 mutant L4 neurons (R31C06-GAL4,dnlg4/UAS-ephrinRNAi,dnlg4) resulted in a frequency of L4 axonal branches invading neighboring columns that was comparable with that of R31C06-GAL4/UAS-ephrinRNAi flies (Figure 7F). Taken together, these results demonstrate that the DNlg4/DNrx interaction promotes DNrx clustering and subsequent ephrin clustering.

DISCUSSION In this study, we showed that DNrx restricts the axon branches of L4 neurons to a single column by regulating ephrin clustering. We identified that the intracellular regions of DNrx and ephrin interact and that this interaction promotes ephrin clustering. We further revealed that a DNlg4/DNrx interaction initiates the clustering of DNrx and subsequently promotes ephrin clustering. The results of our study reveal a novel role of DNrx in promoting ephrin clustering, which restricts L4 axonal branches to a single column. Moreover, we identify a linkage between neurexin and ephrin, two key molecules involved in synaptogenesis and axon targeting, respectively. Ephrin Restricts L4 Axon Branches to a Single Column In Drosophila, the medulla neuropil is patterned into layers and columns. The axons of R7, R8, and five lamina neurons (L1–L5) innervate specific layers of the medulla with topographic columnar restriction of their termini. Layer-specific targeting segregates specific types of information to appropriate layers, while columnar restriction preserves the spatial information from the retina to the medulla target columns (Melnattur and Lee, 2011). Given that each of the five subsets of lamina neurons is packed into a single column, different repulsion molecules might be used for each subset of lamina neurons. In support of this idea, requirements for Dscam2 and N-cadherin in columnar restriction were shown to be cell-type but not layer specific, with Dscam2 identified as a membrane protein for L1 columnar restriction and N-cadherin identified for restricting L5 terminal processes to a single column (Millard et al., 2007; Nern et al., 2008). In this study, we show that ephrin is expressed in L4 neurons and restricts the axon branches of L4 neurons within a column. Consistent with this, ephrin-B1 was shown to control the columnar distribution of cortical pyramidal neurons in mice (Dimidschstein et al., 2013). Columnar restriction is regulated by local repulsions between axon processes (Grueber et al., 2003; Perry and Linden, 1982; Wassle et al., 1981). Contact-mediated repellents can produce an inhibitory effect by inducing the growth cone to collapse and recover its forward growth in an alternative direction, thus diverting axons away from unfavorable destinations. In this study, we show that ephrin levels in L4 axon terminals are positively correlated with axon branch retraction. Consistent with this,

(C) mRNA levels of ephrin in wild-type and dnrxD83/273 mutant heads. Total RNAs were extracted from pupae heads at indicated times APF. Rp49 was used as an internal control. Data are presented as mean ± SEM from three independent experiments. ns, not significant. (D) Ephrin distributions in dnrx mutant and heterozygous clones. Dnrx mutant clones were generated using the MARCM technique. Pupae heads were collected at 55 hr APF and triple-stained with anti-ephrin (red), anti-GFP (green), and 24B10 (blue, for R cell axons) antibodies. Three ephrin staining layers are indicated. Scale bar, 5 mm. Quantification of ephrin levels in each layer in dnrx mutant and heterozygous clones is presented in the lower panel. Six sets of independent data were averaged, and the data are presented as mean ± SEM. ***p < 0.001; ns, not significant. (E) Confocal images showing L4 projections in the flies with ephrin depletion using R31C06-GAL4. Adult heads were stained with anti-GFP (green) and 24B10 (red, for R cell axons) antibodies. The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. Control, n = 23; dnrx, n = 28; L4>ephrinRNAi, n = 15; L4>ephrinRNAi; dnrx, n = 12. ***p < 0.001; ns, not significant. (F) Confocal images showing L4 projections in the flies with Eph depletion using UAS-EphRNAi driven by anatomically restricted GAL4 drivers. The L4 axons that project into neighboring columns are indicated by closed arrows, and the L4 axons that overshoot the M4 layer are indicated by open arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. Control, n = 12; Ln>EphRNAi, n = 13; L4>EphRNAi, n = 10; Repo>EphRNAi, n = 8; R7>EphRNAi, n = 14; ort>EphRNAi, n = 11; cha>EphRNAi, n = 7; otd>EphRNAi, n = 8. ***p < 0.001; ns, not significant. See also Figure S6.

Developmental Cell 41, 1–13, April 10, 2017 9

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Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

Figure 6. DNrx Promotes Ephrin Clustering (A) S2R+ cells were transfected with various expression constructs and immunostained 72 hr post transfection to determine the cellular distributions of ephrin and DNrx. Ephrin expressed alone was diffuse within the cytoplasm. Co-clustering was clearly observed when ephrin was expressed with full-length DNrx, but not when ephrin was expressed with the truncated DNrxD17601813 lacking the ephrin-binding sites. Percentage of overlapping ephrin and DNrx expression in each condition is presented in the lower panel. Data are presented as mean ± SEM from six cells in each condition. ***p < 0.001. (B) Expression and distribution of ephrin in the outer medulla in mutant and rescue flies. Pupae heads were collected at 55 hr APF and double-stained with antiephrin (green) and 24B10 (red, for R cell axons) antibodies. Three ephrin staining layers are indicated. Scale bars, 5 mm. Quantification of ephrin levels in each layer in each genotype is presented in the lower panel. Five flies of each genotype were examined, and more than four sections were checked in each medulla. Data are presented as mean ± SEM. ***p < 0.001; ns, not significant. (C) Confocal images showing medulla terminals of L4 neurons in mutant and rescue flies. Adult heads were stained with anti-GFP (green) and 24B10 (red, for R cell axons) antibodies. The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. dnrx, n = 28; DNrxFL rescue, n = 18; DNrxD17601813 rescue, n = 10. ***p < 0.001; ns, not significant. See also Figure S7.

ephrin-B2 was shown to induce rapid and sustained growthcone collapse and axon retraction in ventrotemporal retinal ganglion cell axons (Petros et al., 2010). Our antibody staining reveals that ephrin is highly expressed at the mid-pupal stage with parallel column arrangement in the outer medulla. Convincingly, the expression pattern of its receptor Eph in the outer medulla is comparable (Dearborn et al., 2002). 10 Developmental Cell 41, 1–13, April 10, 2017

L4 neurons differ from other subsets of lamina neurons and exhibit a unique morphology for terminals and interstitial branches (Nern et al., 2008). Bifunctional activities might be required to achieve the complex morphology of L4 neurons, in which the spread of axonal branches throughout a column is promoted while extra-columnar repulsive processes restrict them to a single column. In this study, we show that knockdown of ephrin

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

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Figure 7. DNlg4 Facilitates DNrx Clustering and Regulates L4 Columnar Restriction (A) Confocal images showing medulla terminals of L4 neurons in full-length DNrx rescue flies (green, upper panel) and the extracellular truncated DNrx rescue flies (green, lower panel). The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. WT (wild-type), n = 22; dnrx, n = 28; DNrxFL rescue, n = 18; DNrxDN rescue, n = 9. ***p < 0.001; ns, not significant. (B) Confocal images showing medulla terminals of L4 neurons in wild-type (green, upper left panel), dnlg4KO10 mutant (green, lower left panel), otd-GAL4/UASDNlg4;dnlg4 rescue (green, upper right panel), and otd-GAL4/UAS-dnlg4RNAi (green, lower right panel) flies. The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. WT, n = 22; dnlg4, n = 15; otd-GAL4/UAS-DNlg4;dnlg4, n = 9; otd-GAL4/UAS-dnlg4RNAi, n = 11. *** p < 0.001. (C) DNlg4 distribution in the developing outer medulla. Pupae heads were collected at 60 hr APF and stained with anti-DNlg4 (green) and 24B10 (red, for R cell axons) antibodies. Scale bars, 10 mm. (D) Expression and distribution of ephrin in the outer medulla in WT and dnlg4KO10 mutants at 55 hr APF. Pupae heads were double-stained with anti-ephrin (green) and 24B10 (red, for R cell axons) antibodies. Scale bars, 5 mm. Quantification of relative ephrin levels in each genotype is presented on the right. Five flies of each genotype were examined, and more than four sections were checked in each medulla. Data are presented as mean ± SEM. ***p < 0.001. (E) Confocal images showing DNrx distribution in the outer medulla of wild-type and dnlg4KO10 mutant flies. Pupae heads were collected at 80 hr APF and stained with anti-DNrx (red) and anti-GFP (green, for L4 axons) antibodies. Scale bars, 10 mm. Quantification of relative DNrx protein levels in each genotype is presented on the right. Five flies of each genotype were examined, and more than four sections were checked in each medulla. Data are presented as mean ± SEM. ***p < 0.001. (F) Confocal images showing medulla terminals of L4 neurons in dnlg4;dnrx double-mutant (green, upper panel) and R31C06-GAL4/UAS-ephrinRNAi;dnlg4 (green, lower panel) flies. The L4 axons that project into neighboring columns are indicated with arrows. Scale bars, 5 mm. Percentages of L4 axons extending to neighboring columns in each genotype are presented on the right. Data are presented as mean ± SEM. dnrx, n = 28; dnlg4, n = 15; dnlg4,dnrx, n = 11; L4>ephrinRNAi, n = 15; L4>ephrinRNAi;dnlg4, n = 10. ns, not significant. See also Figure S8.

Developmental Cell 41, 1–13, April 10, 2017 11

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levels at the mid-pupal stage or impaired ephrin clustering results in defective columnar restriction, suggesting EphB/ephrin signaling plays a repulsive role in this system. However, the molecules responsible for attractive growth throughout the columns in this system need to be further identified. Interestingly, EphB/ ephrin signaling might also serve this function. Studies of the molecular control of dorsal-ventral mapping in chickens and mice have shown that EphB/ephrin-B signaling has either repellent or attractant activities depending on the concentrations of ephrin-B relative to EphB levels (Hindges et al., 2002; McLaughlin et al., 2003).

SUPPLEMENTAL INFORMATION

DNrx Promotes Ephrin Clustering The localization and clustering of ephrins and Eph receptors to specific subcellular positions may be critical for their proper functioning. A previous study showed that ephrins require membrane linkage for clustering and activating Eph receptors on adjacent cells, as soluble versions of ephrins are inactive unless artificially clustered (Davis et al., 1994). The cytoplasmic tails of ephrin-B, which contains PDZ-binding motifs, are able to induce ephrin-B clustering in vivo (Torres et al., 1998). However, the cytoplasmic tail of Drosophila ephrin does not contain a PDZbinding consensus sequence (Bossing and Brand, 2002), indicating that an alternative mechanism induces ephrin clustering in Drosophila. In this study, we show that the intracellular regions of ephrin and DNrx interact to promote ephrin clustering in vivo. Interestingly, unlike ephrin in Drosophila, the cytoplasmic tail of DNrx has PDZ-binding consensus (Li et al., 2007). Our pull-down assay identifies that the ephrin-binding site of DNrx does not overlap with its PDZ-binding consensus. Given that the DNrx molecule associates with several PDZ domain-containing proteins and mediates the assembly of cytoplasmic scaffold complexes (Biederer and Sudhof, 2000; Hata et al., 1996; Koroll et al., 2001), it is reasonable to postulate that DNrx promotes ephrin clustering by assembling cytoplasmic scaffold complexes.

ACKNOWLEDGMENTS

Supplemental Information includes eight figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.devcel.2017.03.004. AUTHOR CONTRIBUTIONS Y.T., L.L., and J.H. designed the experiments; L.L. and Y.T. performed and analyzed the experiments; Xiao-yan Zhang carried out the GST pull-down assay; T.L. performed the Y2H screening; Xinwang Zhang generated dnlg4KO10 mutant flies and the anti-DNlg4 antibody; W.X. provided dnrxD83 and dnlg4KO10 mutant flies as well as anti-DNrx and anti-DNlg4 antibodies; W.X. and J.H. discussed the results; J.H. wrote the paper.

We thank Dr. Manzoor A. Bhat for providing the dnrx273 flies, Dr. Martin Heisenberg for providing L1-GAL4 and L2-GAL4, Dr. Michael B. Reiser for providing split GAL4 flies, Dr. Mark A. Frye for providing Ln-GAL4 flies, Dr. S. Lawrence Zipursky for providing Dac-FLP flies, Dr. Claude Desplan for providing otdGAL4 flies, Dr. Chi-Hon Lee for providing ort-GAL4 and otd-GAL4 flies, the Bloomington Stock Center and Tsinghua Fly Center for providing additional flies, and the members of the Han laboratory for their critical comments on the manuscript. This work was supported by grants from the Ministry of Science and Technology (2014CB942803), the National Natural Science Foundation of China (31471031), the Excellent Youth Foundation of Jiangsu Province of China (BK20140024), the Fundamental Research Funds for the Central Universities (to J. H.), the National Natural Science Foundation of China (31400927 to Y.T.), and the Natural Science Foundation of China (Key Program 30930051 to W.X.). Received: August 10, 2016 Revised: February 3, 2017 Accepted: March 3, 2017 Published: March 30, 2017 REFERENCES Banovic, D., Khorramshahi, O., Owald, D., Wichmann, C., Riedt, T., Fouquet, W., Tian, R., Sigrist, S.J., and Aberle, H. (2010). Drosophila neuroligin 1 promotes growth and postsynaptic differentiation at glutamatergic neuromuscular junctions. Neuron 66, 724–738.

STAR+METHODS

Biederer, T., and Sudhof, T.C. (2000). Mints as adaptors. Direct binding to neurexins and recruitment of munc18. J. Biol. Chem. 275, 39803–39806.

Detailed methods are provided in the online version of this paper and include the following:

Bossing, T., and Brand, A.H. (2002). Dephrin, a transmembrane ephrin with a unique structure, prevents interneuronal axons from exiting the Drosophila embryonic CNS. Development 129, 4205–4218.

d d d

d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Fly Genetics B Generation of Transgenic Flies METHOD DETAILS B Antibodies B Immunostaining B Phenotypic Analysis B Microscopy and Image Analysis B Yeast Two-Hybrid Library Screening B Real-Time RT-PCR B Cell Culture, Transfection, Cell Staining, and Image Analysis B Pulldown Assay B Co-immunoprecipitation QUANTIFICATION AND STATISTICAL ANALYSIS

12 Developmental Cell 41, 1–13, April 10, 2017

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Perry, V.H., and Linden, R. (1982). Evidence for dendritic competition in the developing retina. Nature 297, 683–685. Petros, T.J., Bryson, J.B., and Mason, C. (2010). Ephrin-B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions. Dev. Neurobiol. 70, 781–794. Petrovic, M., and Hummel, T. (2008). Temporal identity in axonal target layer recognition. Nature 456, 800–803. Qiu, X.T., Li, Y.H., Li, H., Yu, Y., and Zhang, Q. (2007). Molecular cloning, mapping, and tissue expression of the porcine cluster of differentiation 14 (CD14) gene. Biochem. Genet. 45, 459–468. Rister, J., Pauls, D., Schnell, B., Ting, C.Y., Lee, C.H., Sinakevitch, I., Morante, J., Strausfeld, N.J., Ito, K., and Heisenberg, M. (2007). Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster. Neuron 56, 155–170. Scheiffele, P. (2003). Cell-cell signaling during synapse formation in the CNS. Annu. Rev. Neurosci. 26, 485–508. Sun, M., Xing, G., Yuan, L., Gan, G., Knight, D., With, S.I., He, C., Han, J., Zeng, X., Fang, M., et al. (2011). Neuroligin 2 is required for synapse development and function at the Drosophila neuromuscular junction. J. Neurosci. 31, 687–699.

Knight, D., Xie, W., and Boulianne, G.L. (2011). Neurexins and neuroligins: recent insights from invertebrates. Mol. Neurobiol. 44, 426–440.

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Lee, T., and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461. Lee, C.H., Herman, T., Clandinin, T.R., Lee, R., and Zipursky, S.L. (2001). N-cadherin regulates target specificity in the Drosophila visual system. Neuron 30, 437–450.

Ting, C.Y., Yonekura, S., Chung, P., Hsu, S.N., Robertson, H.M., Chiba, A., and Lee, C.H. (2005). Drosophila N-cadherin functions in the first stage of the twostage layer-selection process of R7 photoreceptor afferents. Development 132, 953–963.

Li, J., Ashley, J., Budnik, V., and Bhat, M.A. (2007). Crucial role of Drosophila neurexin in proper active zone apposition to postsynaptic densities, synaptic growth, and synaptic transmission. Neuron 55, 741–755.

Ting, C.Y., Herman, T., Yonekura, S., Gao, S., Wang, J., Serpe, M., O’Connor, M.B., Zipursky, S.L., and Lee, C.H. (2007). Tiling of r7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion. Neuron 56, 793–806.

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McGuire, S.E., Mao, Z., and Davis, R.L. (2004). Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci. STKE 2004, pl6. McLaughlin, T., Hindges, R., Yates, P.A., and O’Leary, D.D. (2003). Bifunctional action of ephrin-B1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping. Development 130, 2407–2418. Melnattur, K.V., and Lee, C.H. (2011). Visual circuit assembly in Drosophila. Dev. Neurobiol. 71, 1286–1296. Millard, S.S., Flanagan, J.J., Pappu, K.S., Wu, W., and Zipursky, S.L. (2007). Dscam2 mediates axonal tiling in the Drosophila visual system. Nature 447, 720–724. Missler, M., Zhang, W., Rohlmann, A., Kattenstroth, G., Hammer, R.E., €dhof, T.C. (2003). Alpha-neurexins couple Ca2+ channels Gottmann, K., and Su to synaptic vesicle exocytosis. Nature 423, 939–948. Mountcastle, V.B. (1997). The columnar organization of the neocortex. Brain 120, 701–722. Nern, A., Zhu, Y., and Zipursky, S.L. (2008). Local N-cadherin interactions mediate distinct steps in the targeting of lamina neurons. Neuron 58, 34–41.

Torres, R., Firestein, B.L., Dong, H., Staudinger, J., Olson, E.N., Huganir, R.L., Bredt, D.S., Gale, N.W., and Yancopoulos, G.D. (1998). PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21, 1453–1463. Tuthill, J.C., Nern, A., Holtz, S.L., Rubin, G.M., and Reiser, M.B. (2013). Contributions of the 12 neuron classes in the fly lamina to motion vision. Neuron 79, 128–140. Wassle, H., Peichl, L., and Boycott, B.B. (1981). Dendritic territories of cat retinal ganglion cells. Nature 292, 344–345. Xing, G., Gan, G., Chen, D., Sun, M., Yi, J., Lv, H., Han, J., and Xie, W. (2014). Drosophila neuroligin3 regulates neuromuscular junction development and synaptic differentiation. J. Biol. Chem. 289, 31867–31877. Zeng, X., Sun, M., Liu, L., Chen, F., Wei, L., and Xie, W. (2007). Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila. FEBS Lett. 581, 2509–2516. Zhu, Y., Nern, A., Zipursky, S.L., and Frye, M.A. (2009). Peripheral visual circuits functionally segregate motion and phototaxis behaviors in the fly. Curr. Biol. 19, 613–619.

Developmental Cell 41, 1–13, April 10, 2017 13

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

anti-DNrx rabbit polyclonal antibody

(Tian et al., 2013)

N/A

anti-DNrx mouse monoclonal antibody

(Tian et al., 2013)

N/A

anti-chaoptin mouse monoclonal antibody

DSHB

24B10; RRID:AB_528161

anti-beta-tubulin mouse monoclonal antibody

DSHB

E7; RRID:AB_528499

anti-GFP mouse monoclonal antibody

Roche

Cat#11814460001; RRID:AB_390913

GFP Tag Polyclonal Antibody, Alexa Fluor 488

Invitrogen

Cat#A-21311; RRID:AB_221477

Alexa Fluor 488 goat anti-mouse IgG

Abcam

Cat#ab150113; RRID:AB_2576208

Dylight 550 goat anti-mouse IgG

Abcam

Cat#ab96876; RRID:AB_10679663

Alexa Fluor 647 goat anti-mouse IgG

Abcam

Cat#ab150115

Alexa Fluor 488 donkey anti-rabbit IgG

Abcam

Cat#ab150073; RRID:AB_2636877

Dylight 550 goat anti-rabbit IgG

Abcam

Cat#ab96884; RRID:AB_10680253

anti-syntaxin mouse monoclonal antibody

DSHB

8C3; RRID:AB_528484

anti-DNlg4 rabbit polyclonal antibody

(Li et al., 2013)

N/A

anti-RFP polyclonal antibody

Invitrogen

Cat#R10367; RRID:AB_10563941

Y2HGold Yeast Strain

Clontech

Cat#630489

Y187 Yeast Strain

Clontech

Cat#630489

Antibodies

Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteins Effectene Transfection Reagent

Qiagen

Cat#301425

MBP-EphrinC

This paper

N/A

GST-Nrx1732-1837

This paper

N/A

GST-Nrx1732-1759

This paper

N/A

GST-Nrx1732-1787

This paper

N/A

GST-Nrx1732-1813

This paper

N/A

GST-NrxD1760-1787

This paper

N/A

GST-NrxD1760-1813

This paper

N/A

GST-Nrx1732-1833

This paper

N/A

Amylose Resin

New England Biolabs

E8021S

Glutathione Resin

G-Biosciences

Cat#786310

Pierce Protein A Agarose

Thermo Fisher Scientific

Cat#20333

Critical Commercial Assays The Matchmaker Gold Yeast Two-Hybrid System

Clontech

Cat#630489

One Step SYBR PrimerScriptTM RT-PCR Kit

TAKARA

Cat#RR014A

DGRC

Cat#150; RRID:CVCL_Z831

UAS-dNrxFL

(Tian et al., 2013)

N/A

UAS-dNrxD1760-1813

This paper

N/A

UAS-dNrxDN

(Tian et al., 2013)

N/A

UAS-dnrxRNAi

TsingHua Fly Center

THU0854

UAS-ephrinRNAi

TsingHua Fly Center

THU1520

UAS-ephRNAi

TsingHua Fly Center

THU3972&TH04366.N

UAS-dnlg4RNAi

VDRC

V6792

UAS-mCD8::GFP

Bloomington Stock Center

BL-5137

Experimental Models: Cell Lines S2R+ Experimental Models: Organisms/Strains

(Continued on next page)

e1 Developmental Cell 41, 1–13.e1–e4, April 10, 2017

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

tubulin-Gal80ts

Bloomington Stock Center

BL-7019

repo-Gal4

Bloomington Stock Center

BL-7415

R7-Gal4

Bloomington Stock Center

BL-8603

ort-Gal4

(Ting et al., 2014)

N/A

otd-Gal4

(Ting et al., 2014)

N/A

dnlg4KO10

This paper

N/A

dnlg3KO127

(Xing et al., 2014)

N/A

dnlg2KO70

(Sun et al., 2011)

N/A

Ln-Gal4

(Zhu et al., 2009)

N/A

L1-Gal4

(Rister et al., 2007)

N/A

L2-Gal4

(Rister et al., 2007)

N/A

R64B03AD

(Tuthill et al., 2013)

N/A

R75H07DBD

(Tuthill et al., 2013)

N/A

R31C06-GAL4

Bloomington Stock Center

BL-49883

ap-GAL4

Bloomington Stock Center

BL-3041

6-60 Gal4

(Nern et al., 2008)

N/A

dac-Flp

(Millard et al., 2007)

N/A

LexAop-tdTomato

(Tadros et al., 2016)

N/A

R31C06-LexAp65

(Tadros et al., 2016)

N/A

Ephrin primer 50 -TGCTCTCCACCATGTCTAGT-30

This paper

N/A

Ephrin primer 50 -TATGAACCTGATCGAACTC-3

This paper

N/A

This paper

N/A

This paper

N/A

pGBKT7-nrx1716-1838

(Tian et al., 2013)

N/A

pMal-EphirnC

This paper

N/A

pGEX-nrx1732-1837

This paper

N/A

pGEX-nrx1732-1759

This paper

N/A

pGEX-nrx1732-1787

This paper

N/A

pGEX-nrx1732-1813

This paper

N/A

Oligonucleotides

0

Rp49 primer 5 -AACGTTTACAAATGTGTATTCCGACC-3 Rp49 primer 50 -ATGACCATCCGCCCAGCATACAGG-30

0

Recombinant DNA

D1760-1787

This paper

N/A

pGEX-nrxD1760-1813

This paper

N/A

pGEX-nrx1732-1833

This paper

N/A

pAc5.1 vector

Invitrogen

Cat#V411020

pAc5.1-Ephrin

This paper

N/A

pAc5.1-nrxFL

This paper

N/A

pAc5.1-nrxD1760-1813

This paper

N/A

Zeiss

https://www.zeiss.com/microscopy/int/ downloads/lsm-5-series.html

Image J/Fiji-Coloc2 plugin Fiji software

Image J

https://imagej.nih.gov/ij/plugins/colocalization.html

Microsoft Excel

Microsoft

https://www.microsoft.com/en-us/download/ details.aspx?id=10

pGEX-nrx

Software and Algorithms Zeiss LSM Image Browser

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to the Lead Contact, Junhai Han (junhaihan@seu. edu.cn). Developmental Cell 41, 1–13.e1–e4, April 10, 2017 e2

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

EXPERIMENTAL MODEL AND SUBJECT DETAILS Fly Genetics The flies were maintained on standard medium at 25 C with 60–80% relative humidity. The wild-type flies used in this study were w1118. The Drosophila a-nrx (CG7050) null mutant allele, dnrx273, was obtained from Dr. Manzoor A. Bhat’s laboratory (Li et al., 2007). The other a-nrx null mutant allele, dnrxD83, was generated by p-element imprecise excision using D2-3 as a transposase source according to standard procedures. The detailed experimental process has been described in a previous report (Zeng et al., 2007). To eliminate potential genetic background effects, the recombinant of two out-crossed dnrx null mutant alleles, dnrxD83/dnrx273, was used in this study. The dnlg4 (dnlg4KO10), dnlg3 (dnlg3KO127), and dnlg2 (dnlg2KO70) null mutant alleles were generated using ends-out technology according to standard procedures, and the detailed experimental process has been described in previous reports (Sun et al., 2011; Xing et al., 2014). L1-GAL4 and L2-GAL4 were kindly provided by Martin Heisenberg (Rister et al., 2007). Ln-GAL4 was from Mark A. Frye (Zhu et al., 2009). The GAL4 lines R64B03AD attP40; R75H07DBD attP2, and R21A05AD attP40; R31H09DBD attP2 were from Michael B. Reiser (Tuthill et al., 2013). The ort-GAL4 line was obtained from Chi-Hon Lee, and the otd-GAL4 line was generously provided by Claude Desplan and Chi-Hon Lee (Takemura et al., 2011; Ting et al., 2014). Dac-FLP, 6-60 GAL4, LexAop-tdTomato, and R31C06-LexAp65 lines were obtained from Dr. S. Lawrence Zipursky (Millard et al., 2007; Nern et al., 2008; Tadros et al., 2016). The RNAi lines p[UAS-dnrxRNAi] (THU0854), p[UAS-ephrinRNAi] (THU1520), and p[UAS-EphRNAi] (THU3972 and TH04366.N) were bought from Tsinghua University. Other tool lines used in this work, including R31C06-GAL4 and p{GawB}apmd544 (ap-GAL4) labeling L4 neurons, were ordered from the Bloomington Stock Center. To generate single dnrx mutant L4 and L5 neurons, we used a dac-FLP and labeled the mutant neurons with ap-GAL4 and 6-60 GAL4, respectively. Wild-type MARCM clones were generated with FRT82B. Full genotypes of flies shown in main figures are listed in Table S1. Generation of Transgenic Flies To generate the DNrx transgenes, the full-length DNrx cDNAs (DNrxFL) and the cDNAs that encode either the N-terminal truncation (DNrxDN) or the ephrin-binding-site deletion (DNrxD1760-1813) were sub-cloned into the pUAST vectors and injected into w1118 flies. METHOD DETAILS Antibodies Anti-DNrx antibodies that recognize the extracellular region of DNrx were raised in rabbits and in mice against a purified 63His fusion fragment (aa 15341690) of DNrx protein to generate polyclonal and monoclonal antibodies, respectively. An affinity column generated by coupling the same DNrx fragment to Sepharose 4B was used to purify the antibody. Rabbit anti-DNrx was used in Figures 1A, S1A, and S1B, while mouse anti-DNrx was used in Figures 1B, 1C, 1D, 4D, 6A, 7D, and S1C. Ephrin antiserum was generated in rabbits against a synthetic KLH-conjugated peptide of the intracellular domain of ephrin, and the sequence of the peptide synthesized was CNGMFDQNAGTIEYDR (Genscript). Other antibodies were obtained from DSHB (24B10), Roche (mouse monoclonal antibody [mAb] GFP), Invitrogen (Alexa Fluor 488 rabbit anti-GFP), and Abcam (Alexa Fluor 488 goat anti-mouse IgG, Dylight 550 goat anti-mouse IgG, Alexa Fluor 647 goat anti-mouse IgG, Alexa Fluor 488 donkey anti-rabbit IgG, and Dylight Fluor 550 goat anti-rabbit IgG). Immunostaining Adult fly brains were dissected in phosphate-buffered saline (PBS) and fixed with 4% formaldehyde (PFA) for 20 min at room temperature. Fixed brains were blocked with 5% goat serum in PBS with 0.3% Triton X-100 and were incubated with primary antibodies in PBS with 0.3% Triton X-100 at 4 C overnight. After six washes, brains were incubated with secondary antibodies in PBS with 0.3% Triton X-100 at room temperature for 2-3 h. After six washes, brains were mounted for microscopy in vectashield without DAPI (Vector Laboratories). Primary antibodies were mouse mAb 24B10 (1:30), mouse mAb GFP (1:100), rabbit anti-GFP (1:300), mouse mAb DNrx (1:30), rabbit anti-ephrin (1:30). Secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG (1:100), Dylight 550 goat antimouse IgG (1:100), Alexa Fluor 647 goat anti-mouse IgG (1:100), Alexa Fluor 488 Donkey anti-rabbit IgG (1:100) and Dylight 550 goat anti-rabbit IgG (1:100). For pupae head staining, pupae were pre-fixed with 2% PFA for 10 min at room temperature. Pupae brains were dissected in PBS and fixed with 2%PFAwith 0.3% Triton X-100 at room temperature for 90 min. Primary and secondary antibody incubations were performed as described above. Samples were imaged on an LSM 700 confocal microscope. To compare ephrin and DNrx staining signal in different genotypes, more than five heads for each genotype in each condition were prepared in parallel and stained with the same conditions. All samples were imaged using the same protocol. For quantification staining signal, five flies of each genotype were examined, and more than four sections were checked in each medulla. The measurements were graphed using Microsoft Excel, and data are presented as the averages from examined flies. Phenotypic Analysis The overall patterns of R7 and R8 processes were stained with 24B10 antibody, and R7 and R8 were used as references to estimate layer positions and column boundaries. Z-series of 13-mm optical sections containing L4 and R7/8 axons were stacked. Normal L4 axons are arranged in parallel with R7 and R8 axons within one column. When axonal branches of L4 neurons overlapped with e3 Developmental Cell 41, 1–13.e1–e4, April 10, 2017

Please cite this article in press as: Liu et al., Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering, Developmental Cell (2017), http://dx.doi.org/10.1016/j.devcel.2017.03.004

adjacent R7/R8 axons, it was defined as projecting into a neighboring column. More than eight flies of each genotype were examined, and more than fifteen L4 neurons were checked in each medulla. The measurements were graphed using Microsoft Excel. Percentages of axons extending to neighboring columns were calculated as NE/NT, where NE represents the total number of L4 axons that extend into the neighboring column and NT is the total number of examined L4 axons. Microscopy and Image Analysis Zeiss LSM image files were imported into Image J software. DNrx and 24B10 signals across layers were measured by selecting a region of interest and using the ‘‘plot profile’’ command in Image J (Nern et al., 2008). Ephrin and DNrx signals across columns were measured by drawing the entire columns comprising M1-M5 layers in the outer medulla, and the intensities were calculated as the average intensity of one column. Five flies of each genotype were examined, and more than four sections were checked in each medulla. The measurements were graphed using Microsoft Excel, and data are presented as mean ± SEM from examined flies. Yeast Two-Hybrid Library Screening The screening protocol was performed as previously described (Tian et al., 2013). DNrx encoded by the cDNA (aa 17161838) was sub-cloned into the pGBKT7 vector (Clontech) to generate the bait plasmid (pGBKT7-BD-dnrxc). The plasmids from the positive clones were sequenced and identified with the BLAST program and the FlyBase database. Real-Time RT-PCR Total RNAs were extracted from whole pupae using Trizol reagent (Invitrogen). Real-time RT-PCR was conducted using One Step SYBR Primer Script RT-PCR Kit (TAKARA) with primer pairs for ephrin (50 -TGCTCTCCACCATGTCTAGT-30 /50 -TATGAACCTGATC GAACTC-30 ) and rp49 (50 -AACGTTTACAAATGTGTATTCCGACC-30 /50 -ATGACCATCCGCCCAGCATACAGG-30 ). Rp49 was used as a loading control, and the relative mRNA levels were calculated by setting the ephrin mRNA level in the wild-type at 48 h APF as 100%. Cell Culture, Transfection, Cell Staining, and Image Analysis S2R+ cells were grown at 25 C in Schneider’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells in six-well plates (1 3 106 cells per well) were transferred to poly-L-lysine-coated slides and incubated for 12 h. The cells were transfected using the Effectene transfection reagent (Qiagen) according to the manufacturer’s instructions. We used 200 ng per well of pAC5.1-ephrin or pAC5.1-dnrx. Transfected S2R+ cells were fixed in 4% PFA with 0.3% Triton X-100 for 20 min, and were subsequently blocked in 5% goat serum for 30 min. For staining DNrx on the cell membrane, Transfected S2R+ cells were fixed in 4% PFA without Triton X-100 for 20 min. Primary and secondary antibody incubations were carried out in blocking solution for 2 h at room temperature. Primary antibodies were rabbit anti-ephrin (1:30) and mouse anti-DNrx (1:50). Cells were mounted for microscopy. The stained cells were examined under an LSM 700 confocal microscope. Quantification of the ratio of the DNrx overlapped signal was analyzed by Image J/Fiji-Coloc2 plugin Fiji software. Data are presented as the averages from six cells in each condition. Pulldown Assay All GST fusion proteins and MBP fusion proteins were expressed in Escherichia coli BL21 cells and purified with glutathione-Sepharose (GE Healthcare) and amylose resin (New England Biolabs), respectively. To map the binding site in DNrx, various purified GST-DNrx fusion fragment-coupled beads were incubated with purified MBP-ephrin fusion fragments, which contain the intracellular region of ephrin. Reversely, purified MBP-ephrin fusion fragment-coupled beads were incubated with various purified GST-DNrx fusion fragments. After washing, the elution was analyzed by Western blotting. Co-immunoprecipitation Four hundred fly retinas were dissected and homogenized on ice in 4 mL of PBS with a protease inhibitor containing 1% 3-((3-Cholamidopropyl)-dimethylammonio)-1-propanesulfonate (CHAPS) And 1% triton X-100. Co-immunoprecipitation assays were performed as previously described (Tian et al., 2013). After three washes with PBS containing 0.2% CHAPS and 0.1% Triton X-100, the bound complexes were eluted with 23SDS sample buffer and subjected to SDS-PAGE and Western blotting. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses were performed using Microsoft Excel. Data are presented as means ± standard errors of the means (SEMs). For all statistical analysis, Fisher’s exact probability tests were used to compare different genotypes. Significance was classified as p%0.05.

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