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A global timing mechanism regulates cell-type-specific wiring programmes

Abstract

The assembly of neural circuits is dependent on precise spatiotemporal expression of cell recognition molecules1,2,3,4,5. Factors controlling cell type specificity have been identified6,7,8, but how timing is determined remains unknown. Here we describe induction of a cascade of transcription factors by a steroid hormone (ecdysone) in all fly visual system neurons spanning target recognition and synaptogenesis. We demonstrate through single-cell sequencing that the ecdysone pathway regulates the expression of a common set of targets required for synaptic maturation and cell-type-specific targets enriched for cell-surface proteins regulating wiring specificity. Transcription factors in the cascade regulate the expression of the same wiring genes in complex ways, including activation in one cell type and repression in another. We show that disruption of the ecdysone pathway generates specific defects in dendritic and axonal processes and synaptic connectivity, with the order of transcription factor expression correlating with sequential steps in wiring. We also identify shared targets of a cell-type-specific transcription factor and the ecdysone pathway that regulate specificity. We propose that neurons integrate a global temporal transcriptional module with cell-type-specific transcription factors to generate different cell-type-specific patterns of cell recognition molecules regulating wiring.

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Fig. 1: Dynamic expression of wiring genes and ecdysone pathway TFs.
Fig. 2: Ecdysone pathway TFs control multiple aspects of wiring.
Fig. 3: Common and cell-type-specific targets of the ecdysone pathway.
Fig. 4: Screening of genes co-regulated by EcR and Erm for wiring regulators.

Data availability

All raw sequencing data are available under Gene Expression Omnibus (GEO) accession GSE190714. Gene expression tables for all sequencing experiments are provided in Supplementary Tables 69. Raw data for all other experiments are available on request. Source data are provided with this paper.

Code availability

Code used in this work is available on request.

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Acknowledgements

We thank M. Y. Pecot (Harvard), N. Yamanaka (UC Riverside), D. L. Black (UCLA), E. M. De Robertis (UCLA), L. M. Riddiford (University of Washington) and J. W. Truman (Janelia Research Campus) for helpful discussions. We thank O. Schuldiner (Weizmann Institute of Science) and members of the Zipursky laboratory for feedback on the manuscript and M. Diaz de la Loza for help with figure illustrations. Figure 2 and Extended Data Fig. 3 were designed using resources from Flaticon.com. We would like to specifically acknowledge J. Yoo (Zipursky laboratory) and R. Hodge (Jones laboratory, UCLA) for help with ATAC-seq library preparation and immunostaining. We also thank the BSCRC Sequencing Core (UCLA) and the TCGB core (UCLA) for help with library preparation and sequencing; the BSCRC FACS core (UCLA) and the Witte laboratory (UCLA) for assistance with FACS purification of lamina neurons; and IDRE Statistics Consulting (UCLA) and B. Balliu (UCLA) for assistance with the statistical analysis of data. Reagents provided by O. Akin (UCLA), F. A. Laski (UCLA), H. Wang (Duke-NUS), M. Y. Pecot (Harvard), C. S. Thummel (University of Utah) and G. J. Bashaw (University of Pennsylvania) and fly lines from the Bloomington Drosophila Stock Center were critical for this work. This work was supported by NIH T32-NS048004 Neurobehavioral Genetics Training Grant (S.J.), the Helen Hay Whitney Foundation (S.J.) and a Whitcome Fellowship (Y.L.). S.L.Z. is an investigator of the Howard Hughes Medical Institute.

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S.J., Y.L., Y.Z.K., J.V.-A. and S.L.Z. designed experiments. S.J., Y.L., P.M., S.A.L., J.V.-A. and B.P. acquired data. S.J., Y.L., J.V.-A. and Y.K. analysed the data. S.J., Y.L. and S.L.Z. wrote the manuscript with input from all co-authors.

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Correspondence to S. Lawrence Zipursky.

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Extended data figures and tables

Extended Data Fig. 1 Developmental expression of EcR-pathway TFs and bulk ATAC-Seq data.

a, Images showing immunostaining for EcR-B1, EcR-A, Hr3 and Ftz-f1 at the indicated time points. Scale bar, 50μm. Note: EcR-A positive cells at 36hAPF are glia. b, Comparison of replicates of bulk RNA-Seq of L1 neurons at 40hAPF, 60hAPF and 72hAPF. Values given are Spearman correlation values (see Methods). c, Coverage tracks from L1 bulk RNA-Seq in the EcR locus at 40hAPF, 60hAPF and 72hAPF. EcR-A, EcR-B1 and EcR-B2 transcripts are shown, and EcR-B1 specific exon is highlighted. d, Comparison of replicates of bulk ATAC-Seq data of L1 neurons at 40hAPF, 60hAPF and 72hAPF. Values shown are Spearman correlation values (see Methods). e, Comparison of change of ATAC-seq peak coverage (for regions with dynamic coverage over time) and change in expression of nearest gene. log2(fold change RPKM of nearest gene) vs log2(fold change ATAC-seq peak coverage) between 40hAPF and 60hAPF, and 60hAPF and 72hAPF. r, Pearson’s correlation coefficient. f, Distribution of the top 5000 peaks at each time point between various genomic landmarks. g, p-values (two-sided Hypergeometric test) for enrichment of binding motifs of EcR-usp complex, Hr3 and Nr5a1 (mammalian homolog for Ftz-f1) amongst the following sets of ATAC-Seq peaks: peaks going down from 40hAPF to 60hAPF, peaks going up from 40hAPF to 60hAPF, peaks going down from 60hAPF to 72hAPF, peaks going up from 60hAPF to 72hAPF, and peaks invariant over time. Red: occurrence of motif is higher than expected by chance, blue: occurrence of motif is lower than expected by chance (see Methods).

Extended Data Fig. 2 Analysis of morphology and DIP-β expression with or without EcRDN expression.

a, Morphology of lamina neurons (L1-L5) in wildtype (WT) brains and upon pan-lamina expression (using 9B08 Gal4) of EcRDN, EcRDN + EcR-B1 cDNA or EcR RNAi. Arrowhead in inset points to M3 medulla layer. Note: loss of arborization in M3 with EcRDN is likely due to loss of driver expression in L3 neurons (see Extended Data Fig. 2b). b, Morphology of L3 neurons with or without EcRDN expression using an L3-specific driver (9D03 Gal4). Note, in adults 9D03 Gal4 also labels some L2 neurons. This is not the case during development (see Extended Data Fig. 11f, g) a, b, Arrowhead points to M3 layer in the medulla. Scale bar, 50μm. c, Effect of EcRDN expression on morphology of T4/T5 neurons. Four layers in the lobula plate, a, b, c and d, are marked with red, yellow, blue and pink arrowheads, respectively. Cartoons of one T4 (purple neuron, top) and one T5 neuron (purple neuron, bottom) are shown to highlight wildtype morphology. Scale bar, 20 μm. d, DIP-β mRNA expression in L1-L5 neurons across development with or without pan-lamina expression of EcRDN (from scRNA-Seq based transcriptomic analyses, see Fig. 3, Extended Data Fig. 4, 5). * significant difference between control and EcRDN (change in expression > 2-fold, p-value < 0.05, two-sided Wilcoxon rank sum test). e, Staining using anti-DIP-β antibody at the indicated time points in development with or without pan lamina expression of EcRDN. Inset shows the lamina neuropil. Arrowhead points to proximal lamina neuropil, positive for DIP-β staining at 72hAPF. Note that staining is largely absent from the lamina neuropil (yellow arrowheads). The level of DIP-β RNA is reduced in EcRDN at 48 but rises to near normal levels again at 72 h. We assume that the decrease in RNA leads to a lag in the accumulation of DIP-β protein. Scale bar, 50 μm. f, EcR-usp complex binding motif within the first intron of DIP-β (FDR < 10−7, see Methods). For number of replicates and exact p-values, see Supplementary Table 10.

Extended Data Fig. 3 Analysis of wiring defects in L5 with or without disruption of Ecdysone-pathway TFs.

a, Different L5 arborization defects and their distributions under the given genotypes (see Fig. 2g). All transgenes are expressed using an L5-specific driver (pan-lamina driver is used for data in Fig. 2g). n = number of neurons. **, p-value < 0.001. b, schematic showing the EcRFlpStop allele. FlpStop cassette is inserted in the first common intron of EcR (grey bar shows insertion site, Mi{MIC}EcRMI05320). Cells expressing Gal4, within which a stochastic, heat-shock FLP recombinase-mediated flipping of the cassette occurs, express tdTom. c, Ca2+ response from Tm3 (measured using GCaMP6s) upon optogenetic stimulation of L5 with (blue line) or without (red line) EcRDN expression in L5. GCaMP6s responses were measured in 3 regions of interest (ROI). ROI 1, ROI 2 and ROI 3 span medulla layers M1, M5 and M10 respectively. (WT, 19 animals; EcRDN, 17 animals.) d, Ca2+ response from Dm13 and Dm18 (common LexA driver used yields expression in both Dm13 and Dm18, see Supplementary Table 1) upon optogenetic stimulation of L5 with (blue line) or without (red line) EcRDN expression in L5. ROI 1 spans M1 and measures response from Dm18. ROI 2 spans M5 and measures response from Dm13. (WT, 19 animals; EcRDN, 17 animals.) Note: weak response from Dm18 upon stimulation of L5 irrespective of condition. c, d, Amplitude of relative peak response for each condition is quantified. There is no significant difference between WT and EcRDN for any comparison shown here. For Dm13 response, see Fig. 2j. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively. p-value from two-sided Student’s t-test. For number of replicates and exact p-values, see Supplementary Table 10.

Extended Data Fig. 4 Approach for identification of EcR and Hr3 targets using scRNA-Seq.

a, Scheme for scRNA-Seq based transcriptomic analysis of WT and mutant lamina neurons (see Kurmangaliyev et al11. and methods). GFP vs forward scatter 2-D plot showing criteria used to enrich for lamina neurons by FACS is shown on the right. ‘Cells’ highlighted in purple were excluded despite being GFP+ due to their small size. b, tSNE plots showing WT L1-L5 clusters at 24, 36, 48, 60, 72, 84 and 96 hAPF (Adult). c, Log(expression) of previously identified lamina neuron type-specific genes in L1-L5 clusters identified at each time point over development (see Tan et al32.). d, Comparison of lamina neuron transcriptomes generated by scRNA-Seq in this study and by scRNA-Seq in Kurmangaliyev et al. Values given are Spearman correlation values. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively.

Extended Data Fig. 5 scRNA-Seq-based analysis of WT and EcRDN expressing lamina neurons.

a, tSNE plots showing WT and EcRDN-expressing L1-L5 clusters at 24, 48, 72 and 96 hAPF (Adult). b, Number of genes up or downregulated in EcRDN in L1-L5 neurons. c, Expression of EcR in EcRDN-expressing lamina neurons at 48hAPF normalized to mean expression of EcR in wildtype cells at 48hAPF (done separately for each lamina neuron-type). Red dots, mean of data spread. Increase in EcR expression in EcRDN-expressing cells over wildtype is expected to be due to the expression of the EcRDN transgene. Note the poor induction of EcRDN in L2 neurons. p-value from two-sided Student’s t-test (with Bonferrroni correction for multiple comparisons) are stated in the figure for comparison between L2 and other lamina neuron-types. The difference between EcRDN expression in L2 and L3 neurons is not significant likely due to the low cell numbers of EcRDN-expressing L3 neurons. d, Volcano plots showing significant gene expression changes in L1-L5 neurons throughout development. Red dots: fold change > 2 and p-value < 0.05. e, Log(expression) of lamina neuron-type specific TFs with (blue) or without (red) EcRDN. Note no change in expression of TFs ± EcRDN. f, Log(expression) of TFs in the Ecdysone-pathway in WT (red lines) and EcRDN-expressing (blue lines) L1-L5 neurons. *, p-value < 0.05, fold change > 2. For all statistical tests, see Supplementary Table 7 for number of cells for each cell-type, time-point and condition. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively. d, f, p-value from two-sided Wilcoxon rank sum test.

Extended Data Fig. 6 scRNA-Seq-based analysis of w RNAi and EcR RNAi expressing lamina neurons.

a, tSNE plots showing w RNAi and EcR RNAi-expressing L1-L5 clusters at 24, 48, 72 and 96 hAPF (Adult). b, Number of genes up or downregulated in EcR RNAi in L1-L5 neurons. c, Image showing optic lobe (top) stained using an antibody targeting all EcR isoforms (grey) at 24hAPF. Box with green dotted outline marks the region containing lamina neuron cell-bodies. This region is magnified in bottom two panels. Lamina neurons are labeled in magenta. Scale bar, 50 μm. d, Volcano plots showing significant gene expression changes in L1-L5 neurons throughout development. Red dots: fold change > 2 and p-value < 0.05. e, Log(expression) of lamina neuron-type specific TFs with (blue) or without (red) EcR RNAi. Note no change in expression of TFs ± EcR RNAi. f, Log(expression) of TFs in the Ecdysone-pathway in WT (red lines) and EcR RNAi-expressing (blue lines) L1-L5 neurons. *, p-value < 0.05, fold change > 2. For all statistical tests, see Supplementary Table 8 for number of cells for each cell-type, time-point and condition. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively. d, f, p-value from two-sided Wilcoxon rank sum test. For number of replicates and exact p-values, see Supplementary Table 10.

Extended Data Fig. 7 scRNA-Seq-based analysis of w RNAi and Hr3 RNAi expressing lamina neurons.

a, tSNE plots showing w RNAi and Hr3 RNAi-expressing L1-L5 clusters at 24, 48, 72 and 96 hAPF (Adult). b, Number of genes up or downregulated in Hr3 RNAi in L1-L5 neurons. c, Image showing optic lobe (top) stained using an antibody targeting Hr3 (grey) at 24hAPF. Box with green dotted outline marks the region containing lamina neuron cell-bodies. This region is magnified in bottom two panels. Lamina neurons are labeled in magenta. Scale bar, 50 μm. d, Volcano plots showing significant gene expression changes in L1-L5 neurons throughout development. Red dots: fold change > 2 and p-value <  0.05. e, Log(expression) of lamina neuron-type specific TFs with (blue) or without (red) Hr3 RNAi. Note no change in expression of TFs ± Hr3 RNAi. f, Log(expression) of TFs in the Ecdysone-pathway in WT (red lines) and Hr3 RNAi-expressing (blue lines) L1-L5 neurons. *, p-value < 0.05, fold change > 2. For all statistical tests, see Supplementary Table 9 for number of cells for each cell-type, time-point and condition. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively. d, f, p-value from two-sided Wilcoxon rank sum test. For number of replicates and exact p-values, see Supplementary Table 10.

Extended Data Fig. 8 Comparison of genes affected by EcRDN, EcR RNAi and Hr3 RNAi.

a, Top, Venn diagram showing overlap between genes downregulated by EcRDN and EcR RNAi across all time points and lamina neuron-types. Below, log2(normalized expression in w RNAi/EcR RNAi) vs log2(normalized expression in WT/EcRDN) for L1-L5 neurons throughout development. Correlation coefficient, R, is given for comparisons where p < 0.05. b, Top, Venn diagram showing overlap between genes downregulated by EcRDN and Hr3 RNAi across all time points and lamina neuron-types. Below, log2(normalized expression in w RNAi/Hr3 RNAi) vs log2(normalized expression in WT/EcRDN) for L1-L5 neurons throughout development. Correlation coefficient, R, is given for comparisons where p < 0.05. c, Cell-type variability vs Temporal dynamicity plot for EcR RNAi-affected genes (fold change > 2, p-value < 0.05). Cell-type Specific (blue) and Common (red) targets are shown. Darker colors: genes reduced in EcR RNAi, lighter colors: genes increased in EcR RNAi. d, Cell-type variability vs Temporal dynamicity plot for Hr3 RNAi-affected genes (fold change > 2, p-value < 0.05). Cell-type Specific (blue) and Common (red) targets are shown. Darker colors: genes reduced in Hr3 RNAi, lighter colors: genes increased in Hr3 RNAi. e, Maximum change in expression of Ecdysone-pathway TFs in L1-L5 neurons with EcRDN, EcR RNAi and Hr3 RNAi. Note that EcR RNAi often has weaker effect on TF expression as compared to EcRDN. p-value from two-sided Wilcoxon rank sum test.

Extended Data Fig. 9 Clusters of genes most affected by EcRDN, EcR RNAi and Hr3 RNAi.

All genes expressed in L1-L5 neurons (done separately for each cell-type) were clustered into groups (using k-means clustering) based on their expression dynamics (see Methods). Clusters that show maximum upregulation or downregulation with EcRDN are shown in a – c (clusters are indicated in numbers above each graph). Also shown are clusters unchanged by EcRDN. For each panel: left, light grey lines, relative expression of all genes in the cluster; black line, mean of relative expression of all genes in the cluster. Right, red line, mean relative expression in control; blue line, mean relative expression with perturbation. Shades are SEM. d, examples of dynamic wiring genes that are not affected by EcRDN. d, Plots showing maximum change in expression [log2(WT/EcRDN)] caused by EcRDN vs the temporal dynamicity of the gene (calculated separately for each cell-type). Shown in red are wiring genes. Colored region on the plot represents genes not affected by EcRDN. Note many genes including wiring genes (especially in L1, L3 and L4) with high temporal dynamicity scores that are not affected by EcRDN.

Extended Data Fig. 10 Families of genes affected by Ecdysone-pathway and verification using ex vivo culture of pupal brain.

a, Relative WT expression (left), change in expression with EcRDN (center), and change in expression with Hr3 RNAi (right) shown as heat maps for all genes expressed in L3-L5 neurons belonging to the specified gene categories at 48hAPF. Examples of genes belonging to the Dpr family, Side-Beat family and cell-type specific transcription factors are shown separately. b, Expression of some genes involved in ATP synthesis and vacuolar ATPase biology are shown with and without EcRDN in L1-L5 neurons across development. *, fold change between WT and EcRDN > 2, p-value < 0.05. Note: expression in adults is not significantly affected by EcRDN. p-value from two-sided Wilcoxon rank sum test. c, Left, schematic of experimental setup (see Methods). Briefly, brains are dissected at 22hAPF then incubated for 26h in media ± 20E (20 HydroxyEcdysone, active form of Ecdysone). Right, optic lobes stained for EcR-B1, Hr3 and Ftz-f1 cultured ex vivo ± 20E. Scale bar, 50μm. d, Left, Staining for CARPB and dpr6 reporters (reporter lines, see Methods) in lamina neuron cell-bodies ± 20E. Scale bar, 10μm. Right, CARPB and dpr6 expression with (blue) or without (red) EcRDN. *, fold change between WT and EcRDN > 2, p-value < 0.05, two-sided Wilcoxon rank sum test. Note: dpr6 expression is unchanged in L2 with pan-lamina expression of EcRDN, however staining in brains cultured ex vivo without 20E in the medium shows reduced protein expression in L2 (Bab2 positive cell-bodies). This is consistent with weak activity of the pan-lamina driver (9B08 Gal4) in L2 at 48hAPF leading to ineffective inhibition of the Ecdysone-pathway (see Extended Data Fig. 5c). For number of replicates and exact p-values, see Supplementary Table 10.

Extended Data Fig. 11 Genes expressed in L3 required for R8 wiring and model figure.

a, R8 presynaptic sites (Brp, green) ± EcRDN expression in L3. Right, quantification of Brp puncta number under the conditions shown. Scale bar, 5 μm. (WT, 58 neurons; EcRDN, 47 neurons). b, Total number of presynaptic sites (Brp puncta) in R8 neurons with WT, NetB overexpressing, or EcRDN and NetB-expressing L3 neurons. NetB overexpression is unable to rescue the reduction in R8 presynaptic sites seen with expression of EcRDN in L3 neurons. a, b, p-value (two-sided Student’s t-test) is given. c, d, Distributions of R8 axon terminal depth in w RNAi (red) or with expression of other RNAi (as shown, blue) in L3 neurons. p-values (two-sided Kolmogorov-Smirnov test) are given and number of neurons/ conditions are given. ns = Not significant. c, RNAi against TFs in the Ecdysone-pathway. d, Results from RNAi screen showing genes that did not significantly increase R8 axon depth. Note that nrm RNAi yields a subtle reduction in R8 axon depth. Numbers of neurons/ condition are given. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively. e, Expression of NetA and NetB in L3 with (blue) or without (red) EcRDN expression. *, fold change between WT and EcRDN > 2, p-value < 0.05, two-sided Wilcoxon rank sum test. f, Staining using anti-NetB antibody (magenta) at the indicated times in development. Marker for M6 medulla layer (24B10, grey) and L3 neurons labeled with GFP (green) are shown. g, Staining using anti-NetB antibody (magenta) ± EcRDN expression only in L3. L3 neurons labeled with GFP (green) are shown. f, g, Scale bar, 50μm. Yellow arrowhead, M3 medulla layer. For number of replicates and exact p-values, see Supplementary Table 10. h, Model for temporal regulation of wiring genes. Temporally-regulated TFs (grey triangle) controlled by various external cues such as neural activity, cell-cell interactions and endocrine signals (e.g. Ecdysone) work together with cell-type specific TFs (red and blue proteins). Their combined activity controls the timing and cell-type specificity of wiring gene (such as the ones coding for cell recognition molecules) expression.

Supplementary information

Supplementary Information

This file contains Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Fly strains used in this study.

Supplementary Table 2

Temporal dynamicity and cell type variability scores.

Supplementary Table 3

Wiring genes.

Supplementary Table 4

Gene Ontology and Reactome analyses.

Supplementary Table 5

L1 bulk ATAC-seq data.

Supplementary Table 6

scRNA-seq data for wild-type lamina neurons.

Supplementary Table 7

scRNA-seq data for wild-type versus EcRDN-expressing lamina neurons.

Supplementary Table 8

scRNA-seq data for wild-type versus EcR RNAi-expressing lamina neurons.

Supplementary Table 9

scRNA-seq data for wild-type versus Hr3 RNAi-expressing lamina neurons.

Supplementary Table 10

Number of neurons and animals per condition and exact P values for all comparisons described in this study.

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Jain, S., Lin, Y., Kurmangaliyev, Y.Z. et al. A global timing mechanism regulates cell-type-specific wiring programmes. Nature 603, 112–118 (2022). https://doi.org/10.1038/s41586-022-04418-5

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