Epidermis-Derived Semaphorin Promotes Dendrite Self-Avoidance by Regulating Dendrite-Substrate Adhesion in Drosophila Sensory Neurons

Abstract

Precise patterning of dendritic arbors is critical for the wiring and function of neural circuits. Dendrite-extracellular matrix (ECM) adhesion ensures that the dendrites of Drosophila dendritic arborization (da) sensory neurons are properly restricted in a 2D space, and thereby facilitates contact-mediated dendritic self-avoidance and tiling. However, the mechanisms regulating dendrite-ECM adhesion in vivo are poorly understood. Here, we show that mutations in the semaphorin ligand sema-2b lead to a dramatic increase in self-crossing of dendrites due to defects in dendrite-ECM adhesion, resulting in a failure to confine dendrites to a 2D plane. Furthermore, we find that Sema-2b is secreted from the epidermis and signals through the Plexin B receptor in neighboring neurons. Importantly, we find that Sema-2b/PlexB genetically and physically interacts with TORC2 complex, Tricornered (Trc) kinase, and integrins. These results reveal a novel role for semaphorins in dendrite patterning and illustrate how epidermal-derived cues regulate neural circuit assembly.

Figure 1. Sema-2b, but not Sema-2a loss of function leads to an increase in dendritic self-crossings

(A–F) Dendritic patterns of wild-type (A), sema-2b^f02042 (B), sema-2b^C4 (C), sema-2b^f02042/C4 (D), sema-2a^03021/B65 (E), and sema-2a^P2, 2b^f02042/2ab^A15 (F) class IV da neurons. Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. Wild-type animals are w¹¹¹⁸ carrying one copy of ppk-CD4-tdTomato. (G) Schematic of a contacting crossing (top) and a non-contacting crossing (bottom). In the case of contacting crossings, both dendrites (white bars) are in the same X–Y plane. In the case of non-contacting crossings, one or both dendrites (red bars) detach from ECM (green sheet) and become enclosed by the basal surface of epidermal cells (grey sheet). (H) Quantification of crossing points normalized to total dendritic length in wild-type (n=6), sema-2b^f02042 (n=4), sema-2b^C4 (n=4), sema-2b^f02042/C4 (n=7), sema-2a^03021/B65 (n=4), and sema-2a^P2, 2b^f02042/2ab^A15 (n=4) mutant neurons. White bars represent the quantifications of contacting crossings and red bars represent the quantification of non-contacting crossings. Data are plotted as average ± SEM. ns, not significant, and ***p < 0.001 as assessed by one-way analysis of variance and Dunnett’s test. The comparisons of the total number of crossings are labeled on top of the bars. The comparisons of the contacting crossings and non-contacting crossings are labeled in the white bars and red bars, respectively. (I–J) Dendritic patterns of control (I) and sema-2b^C4 (J) class IV neurons generated with MARCM. Scale bars represent 30 μm. (K) Quantification of total crossing points normalized to total dendritic length in control (n=3) and sema-2b^C4 (n=3) class IV da neurons. Data are plotted as average ± SEM. ns, not significant as assessed by a Student’s t test.

Figure 2. Enclosure of terminal dendrites by epidermal cells in sema-2b mutants

(A–B) Wild-type (A) and sema-2b^f02042/C4 (B) dendritic field imaged at 48 hr AEL. (C–D) Wild-type (C) and sema-2b^f02042/C4 (D) dendritic field imaged at 120 hr AEL. Wild-type animals are w¹¹¹⁸ carrying one copy of ppk-CD4-tdTomato. Dendrites attached to the ECM are labeled in green and enclosed dendrites are labeled in magenta. The arrowheads point to terminal dendrites that are enclosed by epidermal cells. Scale bars represent 30 μm. (E) Quantification of percentages of enclosed dendrites at 48 hr, 72 hr, 96 hr and 120 hr AEL in wildtype and sema-2b^f02042/C4 neurons. Data are plotted as average ± SEM. ***p <0.001 as assessed by a Student’s t test. (F) Percentages of detached dendrites that are terminal dendrites in class IV da neurons in wild-type and sema-2b mutants.

Figure 3. Sema-2b is derived from epidermal cells and acts at short range to regulate dendrite adhesion

(A–A′) Anti-Sema-2b immunostaining of a w¹¹¹⁸ wild-type third instar fillet reveals strong expression in epidermal cells. Anti-HRP immunoreactivity labels neurons in the peripheral nervous system. (B–B′) No detectable Sema-2b staining in sema-2b^C4 mutants. Scale bar represents 50 μm. (C–C′) The 2b^L-τGFP reporter labels epidermal cells that express Sema-2b. 2b^L-τGFP animals were immunostained for τGFP and anti-HRP, which labels neurons in the body wall. Some GFP positive epidermal cells are indicated by white arrowheads. (D–D′) The 2b-τMyc reporter labels a subset of Sema-2b-expressing epidermal cells. 2b-τMyc animals were immunostained for τMyc and anti-HRP, which labels neurons in the body wall. Some τMyc positive epidermal cells are indicated by white arrowheads. All the staining images are positioned with the anterior side on the left and dorsal side on the top. (E–I) Dendritic patterns and quantifications of class IV da neurons in sema-2b^f02042/C4 (E, n=7), sema-2b ^f02042/C4 mutant with epidermis-expressing full length Sema-2b (F, n=5), epidermis-expressing membrane tethered Sema-2b (G, n=5), and neuronal-expressing full length Sema-2b (H, n=6). Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. Wild-type animals are w¹¹¹⁸ carrying one copy of ppk-CD4-tdTomato. White bars represent the quantifications of contacting crossings and red bars represent the quantification of non-contacting crossings. Data are plotted as average ± SEM. ns, not significant and ***p<0.001 as assessed by one-way analysis of variance and Bonferroni test. The comparisons of the total number of crossings are labeled on top of the bars. The comparisons of the contacting crossings and non-contacting crossings are labeled in the white bars and red bars, respectively. (J–K) Dendritic patterns of control (J) and sema-2b^C4 (K) epidermal clones (labeled by GFP) generated with MARCM. Class IV da neurons are genetically labeled by one copy of ppk-CD4-td-Tomato. Scale bars represent 10 μm. The boundary between two adjacent epidermal cells is marked by a white dashed line. (L) Quantification of total crossing points among dendrites that are covered by control (n=24) and sema-2b^C4 (n=26) class IV da neurons. Data are plotted as average ± SEM. ns, not significant as assessed by a Student’s t test.

Figure 4. Plexin B is the Sema-2b Receptor that regulates dendrite-ECM adhesion

(A–D) Dendritic patterns of wild-type (A), plexB^KG00878 (B), control RNAi (C) and plexB RNAi (D) class IV da neurons. Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. Wild-type animals are w¹¹¹⁸ carrying one copy of ppk-CD4-tdTomato. (E) Quantification of total crossing points normalized to total dendritic length in wild-type (n=4), plexB^KG00878 (n=6), RNAi control (n=6), and plexB RNAi (n=6) neurons. Data are plotted as average ± SEM. ns, not significant, *p<0.05, ***p < 0.001 as assessed by a Student’s t test. The comparisons of the total number of crossings are labeled on top of the bars. The comparisons of the contacting crossings and non-contacting crossings are labeled in the white bars and red bars, respectively. (F–H) Dendritic patterns of sema-2b^C4/+ (F), plexB^KG00878/+ (G), and sema-2b^C4/plexB^KG00878 (H) class IV da neurons. Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. (I) Quantification of total crossing points normalized to total dendritic length in sema-2b^C4/+ (F), plexB^KG00878/+ (G), and sema-2b^C4/plexB^KG00878 (H) class IV da neurons. White bars represent the quantifications of contacting crossings and red bars represent the quantification of non-contacting crossings. Data are plotted as average ± SEM. ns, not significant and **p < 0.01, ***p<0.001 assessed by one-way analysis of variance and Bonferroni test for all pairs of columns. The comparisons of the total number of crossings in each genotype to sema-2b^C4/+ are labeled on top of the bars. The comparisons of the contacting crossings and non-contacting crossings each genotype to sema-2b^C4/+ are labeled in the white bars and red bars, respectively.

Figure 5. Sema-2b genetically interacts with TORC2 complex and Trc kinase

(A–J) Dendritic patterns of tor^ΔP/+ (A), sema-2b^C4/tor^ΔP (B), rictor^Δ2/+ (C), sema-2b^C4/rictor^Δ2 (D), sin1^e03756/+ (E), sema-2b^C4/sin1^e03756 (F), trc¹/+ (G), sema-2b^C4/trc¹(H), fry¹/+ (I), and sema-2b^C4/fry¹ (J) class IV da neurons. Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. (K) Number of total crossing points normalized to total dendritic length in sema-2b^C4/+ (n=5), tor^ΔP/+ (n=4), sema-2b^C4/tor^ΔP (n=5), rictor^Δ2/+ (n=5), sema-2b^C4/rictor^Δ2 (n=5), sin1^e03756/+ (n=5), sema-2b^C4/sin1^e03756 (n=7), trc¹/+ (n=4), sema-2b^C4/trc¹ (n=4), fry¹/+ (n=5), and sema-2b^C4/fry¹ (n=5) neurons. White bars represent the quantifications of contacting crossings and red bars represent the quantification of contacting crossings. Data are plotted as average ± SEM. **p < 0.01, and ***p < 0.001 as assessed by one-way analysis of variance and Bonferroni test.

Figure 6. Trc kinase acts downstream of Sema-2b/PlexB signaling to promote dendrite adhesion

(A–C) Anti-phospho-Trc (P-T449) immunostaining of a wild-type w¹¹¹⁸ third instar fillet reveals strong labeling in the wild-type class IV da neuron (A). Anti-phospho-TrcT449 shows weak labeling in sema-2b^C4 (B) and plexB^KG00878 (C) mutant class IV da neurons. Scale bar represents 50 μm. Class IV da neurons are genetically labeled by ppk-CD4-td-Tomato. All the staining images are positioned with the anterior side on the left and dorsal side on the top. (D) Quantification of the level of P-T449 normalized to ppk-CD4-td-Tomato in the dendrites of the class IV da neurons in the wild-type, sema-2b^C4, and plexB^KG00878 animals. ns, not significant, **p < 0.01, and ***p < 0.001 as assessed by one-way analysis of variance and Bonferroni test for all pairs of columns. (E–G) Dendritic patterns of sema-2b^f02042/C4 mutants expressing wild-type Trc (E), phosphorylation-site mutated Trc (S292AT449A) (F), and myristoylated Trc (Myr-Trc) (G) in class IV da neurons. Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. (H) Quantification of total crossing points normalized to total dendritic length in sema-2b^f02042/C4 mutants expressing wild-type Trc (n=5), Trc^S292AT449A (n=5), and Myr-Trc (n=5) in class IV da neurons. Data are plotted as average ± SEM. *p < 0.05 and **p <0.01 as assessed by a one-way analysis of variance and Dunnett’s test.

Figure 7. Integrins overexpression suppresses dendrite crossing defects in sema-2b mutant

(A–B) Dendritic patterns of class IV da neurons in sema-2b^f02042/C4 mutants (A) and sema-2b^f02042/C4 mutants with integrins (UAS-mys and UAS-mew) overexpressed in class IV da neurons (B). Dendritic crossings are indicated by blue arrowheads. Scale bars represent 30 μm. (C) Quantifications of crossing points in wild-type (n=6), sema-2b^f02042/C4 (n=5) class IV da neurons and sema-2b^f02042/C4 with integrins overexpressed in class IV da neurons (n=4). Data are plotted as average ± SEM. ns, not significant and ***p<0.001 as assessed by one-way analysis of variance and Bonferroni test for all pairs of columns. The comparisons of the total number of crossings in each genotype and wild-type animals are labeled on top of the bars. The comparisons of the contacting crossings and non-contacting crossings are labeled in the white bars and red bars, respectively. (D) Colocalization of Mys-FLAG and Myc-PlexB in dendrites of class IV da neurons. Mys-FLAG and Myc-PlexB are co-expressed by ppk-Gal4 driver. Class IV da neurons are labeled by ppk-CD4-td-GFP. Scale bar represents 20 m. The line plots of two terminal dendrites are show in D’ and D”.

Figure 8. Mys, a β subunit of integrin, associates with PlexB

(A–B) Western blot showing co-immunoprecipitation of PlexB and Mys. Lysates from S2 cells expressing Mys-FLAG with Myc-PlexB or Dscam-Myc as depicted were incubated with anti-Myc antibody (A) or FLAG antibody bound beads (B). Immunoprecipitates were probed for the presence of Mys-FLAG (A), Myc-PlexB or Dscam-Myc (B). (C) Western blot revealing the extracellular region of Mys is required for the association between Mys and PlexB. Lysates from S2 cells expressing either FLAG-Mys, FLAG-MysEctoTM, FLAG-MysEndoTM, and Myc-PlexB as depicted were incubated with Myc antibody. Immunoprecipitates were probed for the presence of FLAG-Mys, FLAG-MysEctoTM and FLAG-MysEndoTM using anti-FLAG antibody. (D–F) PlexB and Mys interaction detected by in situ PLA on the surface of S2 cells. (D) Diagram showing the principles of in situ PLA: if the two membrane proteins (Mys and PlexB) are within 40nm of each other, the secondary antibodies conjugated with oligonucleotides will be joined together and fluorescently labeled oligonucleotides will then be added to the site by a rolling circle amplification. Both Myc-PlexB and FLAG-Mys are tagged on the extracellular regions. (E) PLA signals in each condition. The interactions were visualized as red fluorescent dots and membrane GFP marked the transfected cells. A cartoon showing the position of the focal plane is shown on the lower left of the panel. (F) Quantification of total PLA intensity per GFP positive cell (n=40 in each condition). Data are plotted as average ± SEM. ***p<0.001 as assessed by one-way analysis of variance and Bonferroni test for all pairs of columns. Scale bars represent 10 μm. A.u. arbitrary unit. (G–I) PlexB and Mys interaction detected by in situ PLA in dendrites of class IV da neurons. PLA signals in a neuron that co-expresses FLAG-Mys and Myc-PlexB are shown in (G). PLA signals in a neuron that co-expresses Mys-FLAG and Myc-PlexB are shown in (H). (I) Quantification of the number of PLA dots normalized to the dendritic length in each genotype. Data are plotted as average ± SEM. ***p<0.001 as assessed by a Student’s t-test. Staining images are positioned with the anterior side on the left and dorsal side on the top. Scale bars represent 10 μm. (J) Diagram of a cross section of the ECM (green), epidermis (blue) and dendrites (red). Sema-2b ligands are produced and secreted by epidermal cells and diffuse throughout the whole dendritic field of class IV da neurons. Sema-2b signals through PlexB receptors in the neurons to regulate dendrite-ECM adhesion. (K) A model of the downstream molecular mechanisms of how Sema-2b/PlexB signaling regulates dendrite-ECM adhesion through the activation of Trc kinase and the binding of integrins to laminin proteins in the ECM.