Adhesive but not signaling activity of Drosophila N-cadherin is essential for target selection of photoreceptor afferents

Abstract

Drosophila N-cadherin (CadN) is an evolutionarily conserved, atypical classical cadherin, which has a large complex extracellular domain and a catenin-binding cytoplasmic domain. We have previously shown that CadN regulates target selection of R7 photoreceptor axons. To determine the functional domains of CadN, we conducted a structure-function analysis focusing on its in vitro adhesive activity and in vivo function in R7 growth cones. We found that the cytoplasmic domain of CadN is largely dispensable for the targeting of R7 growth cones, and it is not essential for mediating homophilic interaction in cultured cells. Instead, the cytoplasmic domain of CadN is required for maintaining proper growth cone morphology. Domain swapping with the extracellular domain of CadN2, a related but non-adhesive cadherin, revealed that the CadN extracellular domain is required for both adhesive activity and R7 targeting. Using a target-mosaic system, we generated CadN mutant clones in the optic lobe and examined the target-selection of genetically wild-type R7 growth cones to CadN mutant target neurons. We found that CadN, but neither LAR nor Liprin-alpha, is required in the medulla neurons for R7 growth cones to select the correct medulla layer. Together, these data suggest that CadN mediates homophilic adhesive interactions between R7 growth cones and medulla neurons to regulate layer-specific target selection.

Figure 1

The intracellular domain of CadN is required for proper growth cone morphology, but is largely dispensable for R7 target selection. R7 target selection was assessed in CadN mutant R7 clones with and without various transgene rescues. Single R7 cells, homozygous of a wild-type FRT40 chromosome (A-A””’) or CadN mutant chromosomes (B-B””’), were generated using GMR-Flp-mediated mitotic recombination, and labeled with mCD8-GFP or synb-GFP (green) using the MARCM system (A-B”’ or A””-B””’, respectively). All R-cell axons were visualized with MAb24B10 (red). Full-length CadN or CadN with various mutations were expressed in the CadN mutant R7 neurons to assess their ability to rescue R7 phenotypes (C-G””’). Layer-specific targeting and growth cone morphology were assessed at 17% APF (A-G’), 35% APF (A”-G’”) and at the adult stage (A””-G””’). (A-A’) At 17% APF, newly differentiated photoreceptor neurons project axons into the anterior edge (to the left) of the medulla neuropil. The wild-type R7 growth cones follow the pioneering R8 axons to reach the R7-temporary layer (arrow), just beneath the R8 growth cones. The lamina growth cones (not shown) then project between R7 and R8 growth cones separating them into two layers (arrow head). (A”-A’”’’) At 35% APF and adult stage, the wild-type R7 and R8 growth cones form two separate layers (as indicated). (B, B’) At 17% APF, approximately 21% CadN mutant R7 axons (arrows) fail to reach the R7-temporary layers; instead, they expand their growth cones incorrectly at the R8-temporary layer or between the R7- and R8-temporary layers. (B”, B’”) At 35% APF, about 55% of CadN mutant R7 axons terminate at incorrect layers. In addition, 63% and 19% of CadN mutant R7 growth cones fail to expand (arrowheads) at 17% and 35% APF, respectively. (B””, B””’) At the adult stage, essentially all CadN mutant R7 axons mistarget to the R8 layers or the layer between the R7 and R8 layers. (C-C””’) Expressing full-length CadN in CadN mutant R7s fully rescues targeting and growth cone morphological defects (arrows). (D-E”’) Expressing CadN with a deletion of the entire juxtamembrane domain (CadN-Δjuxta; D-D””’) or with a triple alanine mutation in the juxtamembrane domain (CadN-AAA; E-E’”) in CadN mutant R7s completely rescues the targeting phenotypes (arrows) albeit some (~20%) show minor morphological defects. (F-G””’) CadN mutant R7s expressing CadN with a deletion of the β-catenin binding site (CadN-Δβcat; F-F””’), or a deletion of the entire cytoplasmic domain (CadN-Δcyto; G-G””’), target correctly at the R7-temporary layer at 17% APF (arrows; F, F’, G, G’). However, at 35% APF, some (12% and 16%) of the R7 growth cones retract to the distal layers, with ~20% of them exhibiting morphological defects (arrows; F”, F’”, G”, G’”). In the adults, 31% and 36% of these rescued R7 growth cones retract to the distal layers (arrows; F””, F””’, G””, G””’). The presumptive R7- and R8-temporary layers are indicated by dotted lines in (A’-G’). (A’-G’, A”’-G”’ and A””’-G””’) High-magnification views of A-G, A”-G” and A””-G””, respectively. Scale bars: in A, 30 μm for A-G, A”-G” and A””-G””; in A’, 10 μm for A’-G’, A”’-G”’ and A””’-G””’.

Figure 2. Exon organization of the Drosophila CadN2 gene

(A) The Drosophila genome contains a CadN–homologous gene, CadN2, which is located next to the CadN gene in a head-to-tail orientation. (B) The CadN2 gene contains 14 exons that span approximately 40 Kb of the genomic DNA. Complex alternative splicing occurs in exons 1, 2, and 11. As a result of the alternative use of exons 1a or 1bcd/2, mature CadN2 mRNAs contain variable 5’ sequences as well as different 5’ splice sites of exon 1bcd. Exons 1a and 2 encode two different signal peptide sequences. Alternative use of the exon 11a,b and its 5’ splice site results in three types of transcripts: (i) one that contains the exon 11a and encodes a receptor form; (ii) another that misses the 5’ splice site of the exon 11a to terminates shortly afterward and encodes a truncated form; and (iii) the last that omits the exon 11 and encodes a secreted form. Constant exons are shown as red boxes and alternative exons as green, blue, or brown boxes. SP: signal peptide; CA: cadherin repeat; NC: noncordate domain; EGF: EGF-like calcium-binding repeat; LamG: Laminin-G-like domain; TM: transmembrane domain; JX: juxtamembrane region; βcat: β-catenin binding region. The domain structure of CadN isoforms is shown for comparison. Variable regions in the cadherin and transmembrane domains of CadN are colored in light blue. (C) Western blot analysis of CadN2 protein expressed in S2 cells. Extracts of S2 cells expressing GFP (lane 1), the myc-tagged CadN2 receptor forms (lanes 2), and the myc-tagged CadN2 truncated forms (lane 3) were analyzed by Western blot and probed with anti-myc antibody. A myc tag was used to mark the C-termini of CadN2 proteins. Doublets of ~220 kDa (lane 2) and ~200 kDa (lane 3) detected by anti-myc corresponds to the receptor (predicted molecular weight ~200 kDa) and truncated forms of CadN2 protein (predicted molecular weight ~180 kDa) with variable glycosylation, respectively. M: molecular mass markers (in kDa). (D) Confocal image of the S2-adhesion cells expressing the myc-tagged CadN2 receptor form and a GFP marker. CadN2 protein and GFP were visualized using anti-myc (red) and anti-GFP antibodies (green), respectively. Note that CadN2 protein was detected on the cell surface and the cell processes. In the figure, two S2 cells expressing only GFP were not labeled by anti-myc antibody. Scale bar: 10 μm.

Figure 3

CadN2 is partly redundant to CadN in R7 target selection. Mosaic CadN CadN2 mutant R7 cells were generated using GMR-Flp and labeled with mCD8-GFP or synb-GFP (green) using the MARCM system. All R-cell axons were visualized with MAb24B10 (red). (A, A’) At 17% APF, approximately 41% of the CadN CadN2 double mutant R7 axons (arrows) terminated incorrectly at the R8-temporary layer or between the R7- and R8-temporary layers. (A’’, A’”) At 35% APF, about 53% of the CadN CadN2 double mutant growth cones terminated at incorrect layers and exhibited collapsed morphology (arrowheads), as seen in CadN mutant R7 growth cones. (B, B’) In the adult, essentially all of the CadN CadN2 double mutant axons (arrows) terminated incorrectly at the R8 layer. In addition, approximately 20% of the CadN CadN2 double mutant R7 axons exhibited novel phenotypes that were not seen in CadN mutants ( supplementary Figures 1 B and C). (C, C’) Expressing CadN-Δcyto in CadN CadN2 double mutant R7 partially rescued the mistargeting phenotypes (approximately 38% mistargeting in adults). (D-D’”) Expressing CadN2-CadN chimera protein in CadN mutant R7s exacerbated R7 targeting and growth cone morphological defects. Approximately twice as many R7 axons mistargeted and exhibited collapsed growth cones as seen in CadN mutants (arrowheads) at the 17 hr (D, D’) and 35 hr AFP (D”, D”’). The presumptive R7- and R8-temporary layers are indicated by dotted lines in (A’, D’). (A’, A’’’, B’, C’, D’, D’’’) High-magnification views of A, A’’, B, C, D and D”, respectively. Scale bars: in A, 30 μm for B, D, A’’, C and D’’; in A’, 10 μm for B’, D’, A’’’, C’ and D’’’.

Figure 4

CadN extracellular domain is required and sufficient for mediating homophilic interactions in vitro. S2-suspension cells expressing full-length CadN (A), CadN-AAA (B), CadN-Δjuxta (C), CadN-Δβcat (D), CadN-Δcyto (E), CadN2 (F), or CadN2-CadN chimera (G) were assessed for their ability to induce cell aggregation in the presence of 10 mM calcium. A GFP marker (blue) was used to label the transfected S2 cells. The S2 cells expressing full-length CadN (A) or cytoplasmic mutants (B-E) form cell clusters. In contrast, the S2 cells expressing CadN2 or CadN2-CadN chimera are dispersed (F, G). (H) A bar chart of the size of the cell aggregates formed by S2 cells expressing full-length CadN (blue bars) and CadN-Δcyto (red bars) in the presence of 5 (stripe bars) or 10 (solid bars) mM of calcium. Cell aggregates are divided into five categories according to the number of cells in each aggregate. X-axis: cell-aggregate size (25-50, 50-100, 100-150, 150-200, and greater than 200 cells per cell cluster). Y-axis: the total number of cells that form a given range of cell cluster size. Bars: means of three independent results. Error bars: standard error. Scale bar in A, 100 μm.

Figure 5

CadN is required in the optic lobe for R7 and R8 layer-specific targeting. Approximately 40% of the medulla neurons were rendered homozygous of a wild-type FRT40 (A-A”’) or CadN mutant (B-B”’) or CadN CadN2 double mutant (C-C”’) chromosomes using the ELF system (see Materials and methods for details). The mosaic tissues were marked by the loss of GFP signal. The layer-specific targeting of R7 and R8 axons were assessed in adult animals, using PanR7-Gal4, UAS-mCD8-GFP (green) (AC’) and Rh5-Gal4, UAS-lacZ (blue)(A”-C”’), respectively. MAb24B10 (red) was used to label all R-cell axons. (A-A”’) In the wild-type control, both R7 and R8 axons targeted to correct layers (arrow). (B-B”’) CadN medulla-mosaic animals exhibited severe retinotopic mapping defects: the R-cell axons frequently crossed over one another with some of them projecting further into the proximal medulla neuropil. Some R7 axons terminated incorrectly in the R8 layer (arrow, B’); many R8 axons projected deeper into the R7 layer (arrows, B”’) or the layers below (double arrows, B”’). In addition, the R7 and R8 axonal termini exhibited abnormal morphologies with some R-cell axons extending processes into proximal layers (double arrows). (C-C”’) Removing both CadN and CadN2 in the medulla neurons resulted in seemingly severer phenotypes. A’-C’ and A”’-C”’ are high magnification views of A-C and A”-C”, respectively. Scale bars: in A, 30 μm for B-C, and A”-C”; in A’, 10 μm for B’-C’ and A”’-C”’.

Figure 6

CadN is required in medulla neurons for R7 targeting in the first layer-selection stage. Optic lobes were rendered wild-type (A-A”’), CadN (B-B”’), or CadN CadN2 double mutant (C-C”’) using the ELF system; the mosaic neurons were marked by the loss of GFP signal. (A-C’) The initial projections of R7 axons into the R7-temporary layer were assessed at 17% APF. R7 axons were labeled using PM181-LacZ (grey) and R-cell axons were visualized with MAb24B10 (red). (B, B’ and C, C’) In the CadN or CadN CadN2 target-mosaic animals, genetically wild-type R7 axons projected into the medulla neuropil, composed of processes of mutant medulla neurons. Unlike in wild-type (A-A’), many of the CadN mutant R7s axons (33.7 %) fail to project past R8 growth cones (arrows). (B”, B”’ and C”, C”’) At 37% APF, R7 and R8 axons exhibit severe topographic mapping and layer-specific targeting defects (arrows) in the CadN or CadN CadN2 mutant target-mosaic animals. Moreover, some R-cell axons extend processes to deeper layers (arrowheads). A’-C’ and A”’-C”’ are high magnification view of A-C and A”-C”, respectively. The presumptive R7- and R8-temporary layers are indicated by dotted lines in (A’-C’). Scale bars: in A, 30 μm for B-C and A”-C”; in A’, 10 μm for B’-C’ and A”’-C”’.

Figure 7

A schematic model for CadN’s function in R7 target selection. CadN mediates adhesive interactions between R7 growth cones and neurites of the medulla neurons during the first target selection stage. The CadN cytoplasmic domain interacts with α/β-catenins to regulate growth cone morphology. LAR and Liprin-α function in R7 growth cones, presumably to regulate actin-cytoskeleton for R7 target selection.