Modular transcriptional programs separately define axon and dendrite connectivity
- Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, United States
- University of California, Los Angeles, United States
Figures
Figure 1 with 1 supplement
Single-cell sequencing reveals eight transcriptionally distinct populations of T4/T5 neurons.
(A) Common morphology of a T4/T5 neuron, with axon and dendrite wiring pattern variations in parentheses. (B) Arrangement of the eight T4/T5 subtypes in the optic lobe. Each subtype is defined by a …
Figure 1—figure supplement 1
T4/T5 neurons robustly cluster into eight transcriptionally distinct populations (48 hr APF).
(A) Principal component analysis (PCA). (B) Independent component analysis (ICA). Distributions of cells along eight principal components (PCs) and eight independent components (ICs). (C) tSNE plots …
Figure 2
Three primary axes of transcriptional diversity define eight T4/T5 populations.
(A) Three independent components (ICs, henceforth Axis 1, 2, 3) separate cells into approximate halves. Histograms (bottom) and 1-D scatterplots (top) show the distributions of cells along each …
Figure 3 with 1 supplement
Primary axes of transcriptional diversity define groups of T4/T5 subtypes with shared wiring patterns.
(A–A”) 1-D scatterplots show distribution of cells along Axis 1, 2, and 3 for each cluster. Normalized expression levels are indicated by color, as in scale. (B–B”) In vivo expression of marker …
Figure 3—figure supplement 1
Expression patterns of known marker genes for a/b and c/d subtypes along Axis 1 at 48 hr APF.
See legend of Figure 3 for details.
Figure 4 with 1 supplement
Transcriptional program of a single T4/T5 subtype.
Pairwise comparisons between T4a and other subtypes (‘one versus one’, see Materials and methods) that differ by either axonal outputs (A–C), or dendritic inputs (D). For each comparison, insets …
Figure 4—figure supplement 1
Pairwise comparisons between T4a and subtypes that differ by both axonal outputs and dendritic inputs.
Top 20 DEGs are shown for each comparison. See legend of Figure 4 for details.
Figure 5 with 1 supplement
Dynamics of T4/T5 transcriptional programs during development.
Distributions of normalized expression levels of TFs (A) and selected families of CSPs (B-D) at 24 hr and 48 hr APF. Distributions for each subtype are color-coded as in Figure 1. See also Figure …
Figure 5—figure supplement 1
Single-cell profiling of T4/T5 neurons at 24 hr APF.
Unsupervised analysis revealed eight transcriptionally distinct populations. (A) tSNE plot of 3833 single-cell transcriptomes. (B). Distribution of cells along three primary axes of transcriptional …
Figure 6 with 2 supplements
grn controls sublamination of T4/T5 axons into inner and outer LoP layers.
(A) Schematic of grn+ (red) T4/T5 subtypes in wild-type optic lobe. grn expression defines inner LoP layer subtypes. See also Figure 6—figure supplement 1. (B) grn RNAi and grn overexpression in all …
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Figure 6—source data 1
Cell number quantification data for Figure 6D–E and Figure 6—figure supplement 2.
- https://doi.org/10.7554/eLife.50822.014
Figure 6—figure supplement 1
Sequential lamination of T4/T5 axons and four LoP layers.
23G12-Gal4 drives membrane localized RFP (grey) and nuclear localized GFP (green) in all T4/T5 neurons throughout pupal development.
Figure 6—figure supplement 2
grn RNAi and grn overexpression cause significant loss of T4/T5 neurons between 24 and 48 hr APF.
Statistical significance assessed by one-way ANOVA with Dunnett’s multiple comparison test (**p<0.01).
Figure 7 with 2 supplements
Modular transcription factor codes define eight T4/T5 subtypes.
A common T4/T5 regulatory program is defined by TFs expressed in all subtypes (Davie et al., 2018; Konstantinides et al., 2018; Contreras et al., 2018; Schilling et al., 2019). This program is …
Figure 7—figure supplement 1
Expression patterns of TFs at 24 hr APF.
See Figure 7 legend for details.
Figure 7—figure supplement 2
Expression patterns of subtype-enriched CSPs with cell adhesion domains.
Expression patterns of subtype-enriched CSPs with cell adhesion domains (e.g. Ig and LRR). Expression patterns are shown for 24 hr and 48 hr APF. CSPs are ordered by similarity of their expression …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | MCFO-1 (pBPhsFLP2::PEST;+; UAS-FSF-smGdP::HA_V5_FLAG) | PMID: 25964354 | RRID: BDSC_64085 | Gift from Aljoscha Nern and Gerald Rubin |
| Genetic reagent (D. melanogaster) | 10XUAS-IVS-myr::tdTomato | Bloomington Drosophila Stock Center | RRID: BDSC_32222 | |
| Genetic reagent (D. melanogaster) | 23G12-GAL4 (T4/T5) | Bloomington Drosophila Stock Center | RRID: BDSC_49044 | T4/T5 driver |
| Genetic reagent (D. melanogaster) | 42F06-GAL4 (T4/T5) | Bloomington Drosophila Stock Center | RRID: BDSC_41253 | T4/T5 driver |
| Genetic reagent (D. melanogaster) | 23G12-LexA (T4/T5) | Bloomington Drosophila Stock Center | RRID: BDSC_65044 | T4/T5 driver |
| Genetic reagent (D. melanogaster) | {R59E08-p65ADZp (attP40); R42F06-ZpGdbd (attP2)} (T4/T5 splitGAL4) | PMID: 28384470 | JRC_SS00324 | |
| Genetic reagent (D. melanogaster) | UAS-CD4-tdGFP | Bloomington Drosophila Stock Center | RRID: BDSC_35839 | |
| Genetic reagent (D. melanogaster) | UAS-H2A::GFP | PMID: 26687360 | N/A | Gift from Barret Pfeiffer and Gerald Rubin |
| Genetic reagent (D. melanogaster) | LexAop-myr::tdTomato | PMID: 24462095 | N/A | |
| Genetic reagent (D. melanogaster) | 10XUAS-IVS-myr::GFP | Bloomington Drosophila Stock Center | RRID: BDSC_32197 | |
| Genetic reagent (D. melanogaster) | 10XUAS-IVS-mCD8::RFP | Bloomington Drosophila Stock Center | RRID: BDSC_32219 | |
| Genetic reagent (D. melanogaster) | Mi{PT-GFSTF.1}klg[MI02135-GFSTF.1] | Bloomington Drosophila Stock Center | RRID: BDSC_59787 | |
| Genetic reagent (D. melanogaster) | Mi{PT-GFSTF.1}beat-IV[MI05715-GFSTF.1] | Bloomington Drosophila Stock Center | RRID: BDSC_66506 | |
| Genetic reagent (D. melanogaster) | dpr2-Gal4 | Hugo J. Bellen | N/A | Gift from Hugo J. Bellen |
| Genetic reagent (D. melanogaster) | P{w[+mW.hs]=GawB}grn[05930-GAL4] | Bloomington Drosophila Stock Center | RRID: BDSC_42224 | |
| Genetic reagent (D. melanogaster) | Mi{y[+mDint2]=MIC}beat-VI[MI13252] | Bloomington Drosophila Stock Center | RRID: BDSC_58680 | |
| Genetic reagent (D. melanogaster) | P{y[+t7.7] v[+t1.8]=TRiP.HMS01085}attP2 | Bloomington Drosophila Stock Center | RRID: BDSC_33746 | UAS-grnRNAi |
| Genetic reagent (D. melanogaster) | P{UAS-p35.H}BH1 | Bloomington Drosophila Stock Center | RRID: BDSC_5072 | |
| Genetic reagent (D. melanogaster) | UAS-grn.ORF.3xHA | FlyORF | Stock #: F001916 | |
| Antibody | Chicken polyclonal anti-GFP | Abcam | Cat. #: ab13970 RRID: AB_300798 | IHC (1:1000) |
| Antibody | Rabbit polyclonal anti-dsRed | Clontech | Cat. #: 632496 RRID: AB_10013483 | IHC (1:200) |
| Antibody | Mouse monoclonal anti-Brp | Developmental Studies Hybridoma Bank | Cat. #: nc82 RRID: AB_2314866 | IHC (1:20) |
| Antibody | Mouse monoclonal anti-V5 | Abcam | Cat. #: ab27671 RRID: AB_471093 | IHC (1:300) |
| Antibody | Rabbit polyclonal anti-Dcp-1 | Cell Signalling | Cat. #: 9578 RRID: AB_2721060 | IHC (1:50) |
| Antibody | Goat polyclonal anti-chicken IgY Alexa Fluor 488 | Invitrogen | Cat. #: A11039 RRID: AB_142924 | IHC (1:200) |
| Antibody | Goat polyclonal anti-mouse IgG Alexa Fluor 488 | Invitrogen | Cat. #: A11029 RRID: AB_138404 | IHC (1:500) |
| Antibody | Goat monoclonal anti-rabbit IgG Alexa Fluor 568 | Invitrogen | Cat. #: A11011 RRID: AB_143157 | IHC (1:200) |
| Antibody | Goat polyclonal anti-rat IgG Alexa Fluor 568 | Invitrogen | Cat. #: A11077 RRID: AB_141874 | IHC (1:500) |
| Antibody | Goat oligoclonal anti-rabbit IgG Alexa Fluor 647 | Invitrogen | Cat. #: A27040 RRID: AB_2536101 | IHC (1:200) |
| Antibody | Donkey polyclonal anti-mouse IgG Cy5 | Jackson ImmunoResearch Laboratories | Cat. #: 715-175-150 RRID: AB_2340819 | IHC (1:200) |
| Chemical compound, drug | Papain | Worthington | Cat. #: LK003178 | |
| Chemical compound, drug | Liberase protease | Sigma-Aldrich | Cat. #: 5401119001 | |
| Software, algorithm | Cell Ranger 2.2.0 | https://10xgenomics.com | RRID:SCR_017344 | |
| Software, algorithm | Seurat 2.3.4 | https://satijalab.org/seurat/ | RRID: SCR_016341 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.50822.018
