1. Cell Biology
  2. Developmental Biology
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Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells

  1. Elise Houssin
  2. Mathieu Pinot
  3. Karen Bellec
  4. Roland Le Borgne  Is a corresponding author
  1. Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de Rennes) - UMR 6290, F- 35000, France
  2. Equipe Labellisée Ligue Nationale contre le cancer, United Kingdom
Research Article
Cite this article as: eLife 2021;10:e66659 doi: 10.7554/eLife.66659
7 figures, 1 video, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Distribution of polarity markers and Notch during sensory organ precursor (SOP) cell division.

(A–D’’) Time-lapse imaging of Crumbs (Crb)-GFP (A, A’, n = 22), atypical protein kinase C (aPKC)-GFP (B, B’, n = 10), Bazooka (Baz)-GFP (C, C’, n = 28), and NiGFP (D, D’, n = 25) during SOP cytokinesis. SOPs and their daughter cells are identified by the nuclear markers Histone 2B (H2B)-IRF670 (grey, A and B) and H2B-RFP (grey, D) or by the membrane marker growth-associated protein 43 (GAP43)-IR (magenta, C) expressed under the neur minimal driver. Top views are depicted in A, B, C, and D while the orthogonal views showing the new pIIa-pIIb interface (magenta arrowheads) are depicted in A’, B’, C’, and D’. White dashed lines at t26 (A) and t15 (B) delineate highlight where plot profiles presented in Figure 1—figure supplement 1C' and D' have been performed. Red and blue dots correspond to the anterior pIIb cell and the posterior pIIa cell, respectively. Yellow arrowheads point to NiGFP lateral clusters. Magenta dashed lines delineate the SOP daughters’ cell membrane. 3D schematic representations of the different proteins analysed are depicted in green in A’’, B’’, C’’, and D’’. Apical surface of the pIIb is in red while the apical surface of the pIIa is in blue. The pIIa-pIIb interface is outlined in magenta and the apical surface of neighbouring epidermal cells is outlined in dark grey. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm.

Figure 1—figure supplement 1
Distribution of polarity markers and Notch during sensory organ precursor (SOP) and epidermal cell divisions.

(A–D) Time-lapse imaging of neuroglian (Nrg)-YFP (A, n = 11 SOPs; A’, n = 10 epidermal cells); discs-large (Dlg)-GFP (B, n = 9 SOPs; B’, n = 11 epidermal cells); Crumbs (Crb)-GFP (C, n = 11 epidermal cells), atypical protein kinase C (aPKC)-GFP (D, n = 7 epidermal cells) upon SOP or epidermal cell division. SOPs and their daughter cells are identified by Histone 2B (H2B)-IRF670 expressed under the neur minimal driver (grey). Top views are depicted in upper panels while the orthogonal views showing the new interface (magenta arrowheads for SOP daughters and yellow arrowheads for epidermal cells daughters) are depicted in lower panels. Red and blue dots correspond to pIIb and pIIa daughter cells, respectively. White dots correspond to the daughters of epidermal cells. (C’, D’) Plot profiles representing the fluorescence intensity of Crb-GFP (C’) and aPKC-GFP (D’). Plot profiles have been performed in the SOPs’ daughters along the white dashed lines depicted in Figure 1A and B for the SOPs and along the white dashed lined depicted in (C, D) for the epidermal cells’ daughters. (E) Histogram representing the time of appearance of the indicated GFP markers at the apical interface between epidermal daughters (grey bars) and SOP daughters (green bars). (F, G) Time-lapse imaging of Bazooka (Baz)-GFP (F, n = 31) and NiGFP (G, n = 12) upon epidermal cell division. Top views are depicted in upper panels while the orthogonal views showing the new interface (yellow arrowheads) are depicted in lower panels. White dots correspond to the daughters of epidermal cells. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm.

Dynamics of colocalisation of NiGFP and Bazooka (Baz)-mCherry at pIIa-pIIb interface.

(A–A’) Schematic representation and time-lapse imaging of a dividing sensory organ precursor (SOP) expressing NiGFP (green) adjacent to epidermal cells expressing untagged version of Notch (n = 3). (B–B’) Schematic representation and time-lapse imaging of a dividing SOP expressing untagged version of Notch adjacent to epidermal cells expressing NiGFP (green, n = 5). Dashed and continuous lines represent the plasma membrane of SOP daughters and epidermal cells, respectively. Insets highlighted in red-dashed rectangles in A’ and B’ correspond to the apical and lateral interface at t21. SOPs and their daughter cells are identified by Histone 2B (H2B)-IRF670 expressed under the neur minimal driver (magenta). Clones of cells expressing untagged version of Notch are identified by the presence of the nuclear marker nls-RFP. Red and blue dots correspond to pIIb and pIIa daughter cells, respectively. (C) Localisation of NiGFP (green) together with ubi-Baz-mCherry (magenta) at t20 during SOP cytokinesis (n = 9). White arrowheads point to Baz- and Notch-positive clusters at the apical and lateral interface. Red and blue dots correspond to pIIb and pIIa daughter cells, respectively. (D) Localisation of E-cad-GFP (green) together with Baz-Scarlet (magenta) at t21 during SOP cytokinesis (n = 9). White and yellow arrowheads point to Baz-positive clusters and E-cad-positive clusters at the interface, respectively. Red and blue dots correspond to pIIb and pIIa daughter cells, respectively. (E, E’) Kymographs (E’) generated from high-resolution acquisitions (every 2 s, E) of epidermal-epidermal interface (upper panel, n = 10) or pIIa-pIIb interface (middle and lower panels) (n = 10). NiGFP is in green and ubi-Baz-mCherry is in magenta. Yellow-dashed rectangles highlight the position where the kymographs have been performed. Apical acquisitions have been taken around t12 and lateral acquisitions around t20. On the kymographs, tracks correspond to the movement of the clusters. SOPs and their daughter cells are identified by H2B-IRF670 expressed under the neur minimal driver (grey, C and E). In D, SOPs were identified based on the posterior crescent of Baz in prometaphase. (E’’) Histogram representing the NiGFP/Baz-mCherry colocalisation (Mander’s coefficient) based on kymographs (n = 10 for epidermal, n = 10 for SOP). For epidermal cells, only apical acquisitions have been considered, while apical and lateral acquisitions have been considered for pIIa-pIIb interface. ***p-value ≤ 0.001. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm in A’, B’, C, and D and 1 μm in E’.

Notch loss-of-function does not impair the formation of Bazooka (Baz) clusters along the pIIb-pIIb-like interface.

(A–C) SO lineage analysis using the SO marker Cut (green), the socket marker Su(H) (magenta) and the neuronal marker Elav (blue) in control lineage (A, n = 3 nota), upon silencing of Notch (B, n = 2 nota) or upon degradation of NiGFP with degradFP (C, n = 4 nota). Upon degradation of NiGFP by degradFP (C), 58.5% and 10% of Cut-positive cells are Elav or Su(H) positive, respectively (n = 1045 Cut-positive cells). In a control situation, only 25% of the Cut-positive cells are Elav or Su(H) positive. (D) Localisation of NiGFP (green) together with growth-associated protein 43 (GAP43)-IR (magenta) expressed under the neur minimal driver in control or upon NiGFP degradation by degradFP (n = 19). (E–F) Localisation of Baz-GFP (green in E) and Baz-mScarlet (magenta in F) together with GAP43-IR expressed under the neur minimal driver (grey). Yellow arrowheads point the clusters at the lateral interface. Red dots label pIIb ant pIIb-like cells. (E’–F’) Quantification of the number of Baz-positive lateral clusters at t20/21 in control (E’, n = 10; F’, n = 14) or upon Notch silencing (E’, n = 11) or upon NiGFP degradation by degradFP (F’, n = 16). ns, not statistically significant, p-value ≥ 0.05. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm.

Figure 4 with 2 supplements
Bazooka (Baz) and Delta are required for localisation of Notch in lateral clusters and for Notch activation.

(A–D’’) Time-lapse imaging of NiGFP (green) together with H2B-RFP expressed under the neur minimal driver (magenta) during sensory organ precursor (SOP) cytokinesis in control (A), upon silencing of Baz (B), Delta (C), or both (D). Yellow arrowheads point to NiGFP-positive lateral clusters. Red and blue dots correspond to the pIIb or pIIb-like cells and pIIa cell, respectively. 3D schematic representations of NiGFP localisation (green) along the interface in the different genetic contexts are depicted in A’, B’’, C’’, and D’’. Apical surface of pIIb and pIIb-like cells is in red while the apical surface of the pIIa cellis in blue. The interface between the SOP daughters is outlined in magenta (A’) or in red (B’’, C’’, and D’’) and the apical surface of neighbouring epidermal cells is outlined in dark grey. Quantification of NiGFP fluorescence intensity at the apical interface between SOP daughters are shown in B’, C’, and D’. Control (n = 11) is in green while baz RNAi (n = 11), Delta RNAi (n = 13), and baz, Delta RNAi (n = 14) are in magenta. ns, not statistically significant, p-value > 0.05 and **p-value ≤ 0.01. (E–E’) Quantification of the number (E) and fluorescence intensity (E’) of NiGFP-positive lateral clusters over time in control (green, n = 11 or upon silencing of Baz [n = 11], Delta [n = 13], or both [n = 14] RNAi in magenta). ns, not statistically significant, p-value ≥ 0.05, *p-value < 0.05 and ***p-value ≤ 0.001. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm.

Figure 4—figure supplement 1
NiGFP localisation in bazEH747 mutant clones.

(A–A’) Bazooka (Baz) immunostaining using an anti-Nterm (green, A) or an anti-PDZ antibody (green, A’). bazEH747 mutant clones are identified by the loss of the nuclear marker nls RFP (magenta). Note that the nuclear staining seen with the anti-PDZ antibody in A’ is not specific. (B–B’) Time-lapse imaging of NiGFP (green) in control (B, n = 9) or bazEH747 mutant (B’, n = 10) during sensory organ precursor (SOP) cytokinesis. SOPs and their daughter cells are identified by Histone 2B (H2B)-IRF670 expressed under the neur minimal driver (grey). Yellow arrowheads point to NiGFP-positive lateral clusters. Red and blue dots correspond to the pIIb or pIIb-like cells and pIIa, respectively. (C) Quantification of NiGFP fluorescence intensity at the apical interface between SOP daughters in control (green, n = 9) and in bazEH747 mutant (magenta, n = 10). **p-value ≤ 0.01. (D–D’) Quantification of the number (D) and fluorescence intensity (D’) of NiGFP-positive lateral clusters over time in control (green, n = 8) or in bazEH747 mutant (magenta, n = 10). ns, not statistically significant, p-value > 0.05, *p-value ≤ 0.05 and ***p-value ≤ 0.001. (E–E’) SO lineage analysis using the SO marker Cut (magenta), the socket marker Su(H) (green in E’), or the neuronal marker Elav (green in E) in bazEH747 mutant lineage (n = 76 SOP). bazEH747 mutant clones are identified by the loss of the nuclear marker nls-RFP (blue). The percentage refers to the number of SO. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm.

Figure 4—figure supplement 2
Requirements for Delta and Bazooka (Baz) for Notch activation.

(A–D) SO lineage analysis using the SO marker Cut (blue), the socket marker Su(H) (red), and the neuronal marker Elav (green) in control (A, n = 4 nota), upon silencing of Baz (B, n = 4 nota), Delta (C, n = 2 nota), or both (D, n = 2 nota). (A’–D’) Pictures of adult nota of control (A’) or upon silencing of Baz (B’), Delta (C’), or both (D’), n ≥ 50 for each genotype. (E–F) Efficiency of Baz silencing. Imaging of NiGFP (green) and Baz (magenta) in control (E–F), upon silencing of Baz (E) or upon silencing of Baz and Delta (F). Sensory organ precursors (SOPs) and their daughter cells are identified by Histone 2B (H2B)-RFP expressed under the neur minimal driver (grey). White-dashed lines delineate the pnr-GAL4 domain. Yellow-dashed rectangles in E highlight the high magnifications depicted in panels on the right. Scale bars are 100 μm in A–D’ and 10 μm in E, F.

Neuralized (Neur) localises in and regulates the number of NiGFP/Bazooka (Baz)-positive clusters.

(A) Time-lapse imaging of Neur-GFP (green) together with Baz-Scarlet (red) during sensory organ precursor (SOP) cytokinesis in control (n = 8). Yellow dashed rectangles highlight the high magnifications depicted on the panels on the right. (B) Time-lapse imaging of NiGFP (green) together with Histone 2B (H2B)-RFP expressed under the neur minimal driver (grey) during SOP cytokinesis in neur1F65 (n = 13). Red and blue dots correspond to the pIIb or pIIb-like cells and pIIa, respectively. Clones of mutant cells are identified by the loss of the nuclear marker nls-RFP. (B’) 3D schematic representations of NiGFP localisation (green) along the interface in neur1F65. Apical surface of pIIb and pIIb-like cells is in red. The interface between the SOP daughters is outlined in red and the apical surface of neighbouring epidermal cells is outlined in dark grey. (C) Quantification of NiGFP fluorescence intensity at the apical interface between SOP daughters in control (green, n = 9) and in neur1F65 mutant (magenta, n = 10). *p-value ≤ 0.05. (C–C’’) Quantification of the number (C’) and fluorescence intensity (C’’) of NiGFP-positive lateral clusters over time in control (green, n = 9) or in neur1F65 mutant (magenta, n = 10). *p-value < 0.05, **p-value ≤ 0.01, and ***p-value ≤ 0.001. (D) Localisation of Baz (green) in control (n = 7) neur1F65 mutant (n = 7). Clones of mutant cells are identified by the loss of the nuclear marker nls-RFP (magenta). (D’) Localisation of Baz-mCherry (magenta) together with H2B-IRF670 (grey) expressed under the neur minimal driver in control (n = 23) and upon silencing of Neur (n = 15) during SOP cytokinesis. Pictures show the SOP daughter cells 10 min after the onset of anaphase. White arrowheads point the Baz-positive clusters at the lateral interface. Red and blue dots correspond to the pIIb or pIIb-like cells and pIIa, respectively. (D’’) Quantification of the number of Baz-positive clusters at the lateral interface in control (green, n = 23) or upon silencing of Neur (magenta, n = 15). ***p-value ≤ 0.001. (E) Kymographs generated from high-resolutions acquisition (every 2 s, starting at t12 min) and illustrating the colocalisation between NiGFP (green) and Baz-mCherry (magenta) at the interface of SOP daughters upon silencing of Neur (n = 11). On the kymographs, tracks correspond to the movement of the clusters. (E’) Histogram representing the NiGFP/Baz-mCherry colocalisation (Mander’s coefficient) in apical and lateral clusters based on kymographs in control (green, n = 10) and upon silencing of Neur (magenta, n = 6). Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 and 1 μm for the kymographs.

Numb regulates the number of NiGFP/Bazooka (Baz)-positive clusters.

(A) Time-lapse imaging of Numb-GFP (green) together with Baz-Scarlet (red) during sensory organ precursor (SOP) cytokinesis in control (n = 5). Yellow-dashed rectangles highlight the high magnifications depicted on the panels on the right. (B) Time-lapse imaging of NiGFP (green) together with Histone 2B (H2B)-IRF670 expressed under the neur minimal driver (grey) during SOP cytokinesis upon the silencing of Numb (n = 20). Yellow arrowheads and yellow dashed line point to the lateral clusters. Red and blue dots correspond to the pIIb cells and pIIa or pIIa-like cells, respectively. (B’) 3D schematic representations of NiGFP localisation (green) along the interface upon silencing of Numb. Apical surface of pIIa and pIIa-like cells is in blue. The interface between the SOP daughters is outlined in blue and the apical surface of neighbouring epidermal cells is outlined in dark grey. (C) Quantification of NiGFP fluorescence intensity at the apical interface between SOP daughters in control (green, n = 11) and upon silencing of Numb (magenta, n = 20). *p-value < 0.05 and **p-value ≤ 0.01. (C’–C’’) Quantification of the number (C’) and fluorescence intensity (C’’) of NiGFP-positive lateral clusters over time in control (green, n = 11) or upon silencing of Numb (magenta, n = 20). ***p-value ≤ 0.001. (D–D’’) Localisation of NiGFP (green) together with Baz-Scarlet (magenta) at t14 during SOP cytokinesis in control (D, n = 14), upon silencing of Numb (D’, n = 9) or upon overexpression of Numb (D’’, n = 8). Red and blue dots correspond to the pIIb-like cells and pIIa or pIIa-like cells, respectively. (E) Quantification of the fluorescence intensity of Baz-Scarlet at the apical or lateral interface of control epidermal cells (grey, n = 31), of SOP daughters in the control (green, n = 14), upon silencing of Numb (magenta, n = 9) or upon overexpression of Numb (red, n = 8). ns,not statistically significant, p-value ≥ 0.05, **p-value ≤ 0.01, and ***p-value ≤ 0.001. (F) Kymographs generated from high-resolution acquisition (every 2 s) and illustrating the colocalisation between NiGFP (green) and Baz-mCherry (magenta) at the interface of SOP daughters upon silencing of Numb (n = 14). On the kymographs, tracks correspond to the movement of the clusters. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 and 1 μm for the kymographs.

Figure 7 with 1 supplement
Sanpodo is required for the assembly of NiGFP/Bazooka (Baz) clusters.

(A) Localisation of Sanpodo (Spdo)-GFP (green) together with Baz-Scarlet (magenta) at t21 during sensory organ precursor (SOP) cytokinesis in control (n = 10). Yellow-dashed rectangles highlight the high magnifications depicted on the panels on the right. Yellow arrowheads point to lateral clusters positive for Baz and Spdo. (B) Localisation of Baz (anti-N term, grey) together with the SO marker Cut (green) in control (n = 20 SOP ) or spdoG104 mutant clones (n = 25 SOP). Yellow arrowheads point to Baz-positive clusters at the lateral interface. (C) Time-lapse imaging of NiGFP (green) together with Histone 2B (H2B)-IR (grey) expressed under the neur minimal driver (grey) during SOP cytokinesis in control (n = 14) or in spdoG104 mutant (n = 10). Yellow arrowheads point to NiGFP-positive clusters at the lateral interface while the yellow arrows point to the NiGFP continuous signal along the lateral interface of SOP daughters. spdoG104 mutant clones are identified by the loss of the nuclear marker nls RFP (magenta). Red and blue dots correspond to the pIIb or pIIb-like cells and pIIa cells, respectively. (D) Quantification of NiGFP fluorescence intensity at the apical interface between SOP daughters in control (green, n = 14) and spdoG104 mutant (magenta, n = 10). **p-value ≤ 0.01 and ***p-value ≤ 0.001. (E–E’) Quantification of the t1/2 (E) and the mobile fraction (E’) of NiGFP following fluorescence recovery after photobleaching (FRAP) at the apical interface of SOP daughters in control (black, n = 10) or upon silencing of Spdo (red, n = 11) at t20. **p-value ≤ 0.01 and ***p-value ≤ 0.001. Time is in min. t0 corresponds to the onset of anaphase. Scale bars are 5 μm.

Figure 7—figure supplement 1
Sanpodo is required for the assembly of NiGFP/Bazooka (Baz) clusters.

(A-A’) Localisation of NiGFP (green) together with H2B-IRF670 and growth-associated protein 43 (GAP43) expressed under the neur minimal driver (magenta) at t21 during sensory organ precursor (SOP) cytokinesis upon silencing of Sanpodo (Spdo) (n = 2). Orthogonal views of the interface between SOP daughters are depicted in A’. SOP daughters are highlighted by yellow dashed lines. (B) Time-lapse imaging of NiGFP and the corresponding kymograph after photobleaching at the apical interface between the SOP daughters upon silencing of Spdo (n = 11). The yellow dashed rectangle delineates the photobleached area. (C) Time-lapse imaging of NiGFP (green) together with Baz-Scarlet (magenta) during SOP cytokinesis upon silencing of Spdo (n = 10). Red dots correspond to pIIb and pIIb-like cells. (D–D’) Kymographs generated from high-resolution acquisitions (every 2 s) and illustrating the colocalisation between NiGFP (green) and Baz-mCherry (magenta) at the interface of SOP daughters in control (D, n = 14) and upon silencing of Spdo (D’, n = 7). On the kymographs, tracks correspond to the movement of the clusters. Time is in min except in B where the time is in s. t0 corresponds to the onset of anaphase. Scale bars are 5 and 1 μm for kymographs in B.

Videos

Video 1
3D viewing of the time-lapse of Baz-GFP (green) and GAP43-IR (magenta) from t0 to t18, illustrating the position of the Baz-positive lateral clusters along the pIIa-pIIb interface at t18.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Drosophila melanogaster)w1118Bloomington Drosophila Stock CenterBDSC: 3,605FLYB: FBal0018186;RRID:BDSC_3605
Gene (Drosophila melanogaster)Nrg::YFPP{PTT-GA}NrgG00305Kyoto Stock Center
Morin et al., 2001
FLYB: FBgn0264975;RRID:KSC_110658
Gene (Drosophila melanogaster)neur-H2B::IRThis studyTwo lines generated (IId and IIId)
Gene (Drosophila melanogaster)Crb::GFP (A)Huang et al., 2009FLYB: FBgn0259685Gift from Dr Y Hong
Gene (Drosophila melanogaster)aPKC::GFPBesson et al., 2015FLYB: FBgn0261854Kind gift from Dr F Schweisguth
Gene (Drosophila melanogaster)Baz::GFPP{PTT-GC}bazCC01941Bloomington Drosophila Stock Center
Buszczak et al., 2007
FLYB:FBgn0000163;RRID:BDSC_51572
Gene (Drosophila melanogaster)Baz::mScarletkind gift from Dr J JanuschkeFLYB:FBgn0000163
Gene (Drosophila melanogaster)neur-GAP43::IRThis studyTwo lines generated (IId and IIId)
Gene (Drosophila melanogaster)Dlg::GFPBloomington Drosophila Stock Center
Buszczak et al., 2007
FLYB: FBgn0001624;RRID: BDSC_ 50859
Gene (Drosophila melanogaster)Ubi-p63E-Baz-mCherry (II) and (III)Bosveld et al., 2012Transgenic lines;FLYB:FBgn0000163Kind gift from Dr Y Bellaiche
Gene (Drosophila melanogaster)NiGFPBellec et al., 2018FLYB: FBgn0004647CRISPR line
Gene (Drosophila melanogaster)neur-H2B-RFPGomes et al., 2009Kind gift from Dr F Schweisguth
Gene (Drosophila melanogaster)bazEH747, FRT19A/YKind gift of A WodarzFLYB:FBgn0000163
Gene (Drosophila melanogaster)Ubi-mRFP.nls, w*, hsFLP, FRT19ABloomington Drosophila Stock CenterRRID:BDSC_31418
Gene (Drosophila melanogaster)UAS-Baz dsRNA (III)Bloomington Drosophila Stock CenterRRID:BDSC_35002
Gene (Drosophila melanogaster)UAS-Notch dsRNA (III)Bloomington Drosophila Stock CenterFLYB: FBgn0004647; RRID:BDSC_28981
Gene (Drosophila melanogaster)UAS-Dl dsRNA (III)Bloomington Drosophila Stock CenterFLYB: FBgn0000463; RRID:BDSC_28032
Gene (Drosophila melanogaster)UAS-Nslmb-vhhGFP4 (II)Bloomington Drosophila Stock CenterRRID:BDSC_38422
Gene (Drosophila melanogaster)UAS-Nslmb-vhhGFP4 (III)Bloomington Drosophila Stock CenterRRID:BDSC_38421
Gene (Drosophila melanogaster)pnr-GAL4Bloomington Drosophila Stock CenterFLYB: FBgn0003117; RRID:BDSC_3039Calleja et al., 1996
Gene (Drosophila melanogaster)UAS-Nb dsRNA (III)Bloomington Drosophila Stock CenterFLYB: FBgn0002973; RRID:BDSC_35045
Gene (Drosophila melanogaster)FRT82B, neur1F65Yeh et al., 2000FLYB: FBgn0002932
Gene (Drosophila melanogaster)FRT82B, nls-RFPBloomington Drosophila Stock CenterRRID:BDSC_30555
Gene (Drosophila melanogaster)hsFLPBloomington Drosophila Stock CenterRRID:BDSC_6938
Gene (Drosophila melanogaster)FRT82B, spdoG104O’Connor-Giles and Skeath, 2003FLYB: FBgn0260440
Gene (Drosophila melanogaster)Spdoi-GFP (II)Couturier et al., 2013FLYB: FBgn0260440BAC Rescue, Kind gift from Dr F Schweisguth
Gene (Drosophila melanogaster)UAS-Spdo dsRNA (II)Vienna Drosophila Resource CenterFLYB: FBgn0260440; RRID:VDRC_104092
Gene (Drosophila melanogaster)UAS-Neur dsRNABloomington Drosophila Stock CenterFLYB: FBgn0002932; RRID:BDSC_26023
Gene (Drosophila melanogaster)y, w, PB[y + attP-3B Neur::GFP 22A3]Perez-Mockus et al., 2017FLYB: FBgn0002932BAC Rescue, Kind gift from Dr F Schweisguth
AntibodyAnti-Elav(rat monoclonal)Developmental Studies Hybridoma BankRRID:Rat-Elav-7E8A10IF (1:200)
AntibodyAnti-cut(mouse monoclonal)Developmental Studies Hybridoma BankRRID:2B10IF (1:500)
AntibodyAnti-Su(H)(goat polyclonal)Santa CruzCat# sc15813IF (1:500)
AntibodyAnti-Baz N-term(rabbit polyclonal)Wodarz et al., 1999IF (1:1000),Kind gift from Dr A Wodarz
Antibodyanti-Baz-PDZ (guinea pig)Shahab et al., 2015IF (1:1000),Kind gift from Dr A Wodarz
AntibodyAnti-GFP(goat polyclonal)AbCamCat# Ab5450IF (1:500)
AntibodyCy2-, Cy3- and Cy5-coupled secondary antibodies(donkey anti-goat)Jackson Laboratories705-225-147, 705-165-147, and 705-175-147, respectivelyIF (1:400)
AntibodyCy2-, Cy3- and Cy5-coupled secondary antibodies(goat anti-rabbit)Jackson Laboratories111-225-144, 111-165-144, and 111-175-144, respectivelyIF (1:400)
AntibodyCy2-, Cy3- and Cy5-coupled secondary antibodies(donkey anti-mouse min cross-react with rat)Jackson Laboratories715-225-151, 715-165-151, and 715-175-151, respectivelyIF (1:400)
AntibodyCy2-, Cy3- and Cy5-coupled secondary antibodies(donkey anti-rat min cross-react with mouse)Jackson Laboratories712-225-153, 712-165-153, and 712-175-153, respectivelyIF (1:400)
Software, algorithmImageJ/FIJIOpen source Java image processing programhttps://imagej.nih.gov/ij/
Software, algorithmCountdots macro for FIJIThis study
Software, algorithmExcelMicrosoft Office 2013RRID:Microsoft Excel 2013
Software, algorithmIllustratorAdobe SystemsRRID: Adobe Illustrator CS3
Software, algorithmPrismGraphPadRRID: GrpahpadPrism

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