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Neuronal diversity and convergence in a visual system developmental atlas

Abstract

Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains1,2, the large neuronal diversity of the Drosophila optic lobe3 and its connectome4,5,6 are almost completely characterized. However, a molecular characterization of this neuronal diversity, particularly during development, has been lacking. Here we present insights into brain development through a nearly complete description of the transcriptomic diversity of the optic lobes of Drosophila. We acquired the transcriptome of 275,000 single cells at adult and at five pupal stages, and built a machine-learning framework to assign them to almost 200 cell types at all time points during development. We discovered two large neuronal populations that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signalling throughout development. Moreover, we show that the transcriptomes of neurons that are of the same type but are produced days apart become synchronized shortly after their production. During synaptogenesis we also resolved neuronal subtypes that, although differing greatly in morphology and connectivity, converge to indistinguishable transcriptomic profiles in adults. Our datasets almost completely account for the known neuronal diversity of the Drosophila optic lobes, and serve as a paradigm to understand brain development across species.

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Fig. 1: High-resolution transcriptomic atlases of the optic lobe across development.
Fig. 2: TE neurons demarcate the optic lobe neuropils and undergo apoptosis during late pupal development.
Fig. 3: Transcriptomic synchronization of optic lobe neurons.
Fig. 4: Increased transcriptomic diversity during synaptogenesis.
Fig. 5: Dorsal and ventral visual circuits are partitioned by differential Wnt signalling.

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Data availability

All raw and processed data referenced were uploaded to the GEO with accession number GSE142789. The following publicly accessible datasets were also used: GSE103771, GSE103772 and GSE116969.

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Acknowledgements

We thank all members of the Desplan laboratory, S.-y. Takemura and F. P. Davis for discussions; L. Luo, C. Doe, F. Pinto Teixeira, J. Slabbaert and S. Cordoba for critical reading of the manuscript; and I. Salecker and R. Hiesinger for reagents. The C.D. laboratory is supported by NYSTEM (DOH01-C32604GG), the National Eye Institute (R01 EY017916) and by ADHPG-CGSB1 to the CGSB of the NYU Abu Dhabi Research Institute. M.N.O. is a Leon Levy Neuroscience Fellow. F.S. and Y.-C.C. are supported by New York University (MacCracken Fellowship). S.J. is supported by the Swedish Research Council (Vetenskapsrådet VR grant 2016- 06726). I.H. is supported by a Human Frontier Science Program postdoctoral fellowship (LT000757/2017). J.A.M is supported by the National Eye Institute (F32 F32EY028012). N.K. is supported by the National Eye Institute (K99 EY029356-01).

Author information

Authors and Affiliations

Authors

Contributions

M.N.O., F.S., N.K. and C.D. designed the project. M.N.O., F.S., S.J., I.H., N.B., R.N.E.-D., K.K. and J.A.M. performed the experiments. M.N.O., F.S., S.J., I.H., Y.-C.C., N.B., R.N.E.-D. and K.K. analysed the data. M.N.O., F.S. and S.J. acquired the scRNA-seq data. M.N.O. and F.S. performed all scRNA-seq data analysis and M.N.O., F.S. and C.D. wrote the manuscript. All authors edited the manuscript.

Corresponding authors

Correspondence to Nikolaos Konstantinides or Claude Desplan.

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The authors declare no competing interests.

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Peer review information Nature thanks Stein Aerts and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Batch effect removal and biological relevance of the adult clusters.

a. The proportions of UMIs from mitochondrial genes per cell (n = number of cells in each library, indicated on the right) and the total number of cells passing filters in each of the 15 libraries comprising the adult dataset. Names indicated correspond to the names in the Seurat object provided (Adult.rds, GSE142787). Boxplots display the first, second and third quartiles. Whiskers extend from the box to the highest or lowest values in the 1.5 interquartile range, and outlying data points are represented by a dot. b, Origin of the cells in the final adult clusters, coloured as in a. Green arrows represent clusters for which the unique library distribution can be explained by variable contamination from surrounding tissues (cluster 3 is photoreceptors, 112 is probably Kenyon cells from the central brain) or the number of lamina neuropils dissociated (clusters 107, 108, 109 are lamina neurons). Red arrows indicate clusters that are probably enriched in low quality transcriptomes, as they are enriched in cells from libraries with high number of mitochondrial genes (38, 120, 192) or high number of cells sequenced (102, probably corresponding to multiplets). Brackets indicate glial clusters, some of them enriched in libraries with high number of mitochondrial genes as ambient RNA is more similar to RNA from glial versus neuronal cells (Extended Data Fig. 2). c, Number of clusters obtained with different pairs of clustering parameters. The red rectangle indicates the pair of parameters used. d, Left, Pearson correlation between the average gene expression of the adult dataset clusters (x axis) and the transcriptome of isolated Lawf1 and L1 neurons (Methods). Right, number of isolated neuronal type transcriptomes matching to 1–5 of our adult clusters, for each pair of parameters in c, which we used as a measure of the biological relevance of our clusters. Matching was defined by the presence of a correlation gap greater than 0.05 (Methods). We took into account any correlation gap between the six best-correlated clusters, because similar cell types or overclustering can affect the size of the first correlation gap as illustrated on the left graphs. The red rectangle indicates the pair of parameters used. e, t-SNE visualization of the adult optic lobe single-cell transcriptomes, using 120 principal components calculated on the log-normalized integrated gene expression. Cell colours indicate the cluster they belonged to before we merged artificially split clusters (red circles; Methods). f, Heat map showing scaled log-normalized non-integrated expression of the top20 cluster markers between the merged clusters. Merged clusters had almost indistinguishable gene expression patterns, but often differed by their proportions of UMIs from mitochondrial genes per cell or the expression levels of the genes highlighted in red, which are enriched in the ‘ambient RNA cluster’ 192 (see also Extended Data Fig. 3).

Extended Data Fig. 2 Identification of neuronal and glial clusters.

a, Pearson correlation between the average log-normalized non-integrated expression of the top10 cluster markers of the adult dataset clusters (x axis) and the transcriptome of isolated Repo+ (glial marker) or Elav+ (neuronal marker) populations. LQ, clusters containing a proportion of cells with features of lower-quality transcriptomes; PR, photoreceptor. b, Violin plots of features tending to be higher (proportions of UMIs from mitochondrial genes) or lower (number of UMIs or genes per cell) in low-quality cells48,49. c, Heat map showing the scaled log-normalized non-integrated expression of the top5 cluster markers of the adult dataset. The first 5 neuronal adult clusters (1 to 6, cluster 1 and 2 having been merged) are plotted for reference as they clearly have specific gene expression patterns. Clusters 38, 85, 102 and 120 present much less defined gene expression patterns and probably contain low-quality neuronal transcriptomes (see also Extended Data Fig. 1). Clusters 188 and 189 could be further separated in two groups with different gene expression patterns, as illustrated by the dashed line in the insert. Cluster 191 expresses several markers that are found in no other clusters and probably correspond to neither glia nor optic-lobe neurons. Cluster 192 expresses mainly low levels of glia-specific genes, without specific markers. It probably corresponds to ambient RNA, which would be enriched in RNA from burst glial cells.

Extended Data Fig. 3 Identification of the adult neuronal clusters.

a, Pearson correlation between the average log-normalized non-integrated expression of the top10 cluster markers of the adult dataset clusters (x axis) and the transcriptome of isolated neurons18,20. We represented Dm3, Tm9, T4 and T5 before their split into Dm3a/Dm3b, Tm9v/Tm9d, T4/T5ab and T4/T5cd. When two transcriptomes were published for a given neuronal type, the one presenting the highest correlation gap is displayed in this figure. R1–8, average gene expression of all photoreceptors20. KC, Kenyon cells; cluster 112 therefore probably corresponds to contamination from the central brain. b, Legend as in a but for LC cells. We indicated several matching clusters to highlight the high similarity between the transcriptomes of LC cells, which explains the lower correlation gaps observed for these neurons. c, Left, legend as in a but for Pm3 cells. Right, mixture modelling of Pm3 markers19 (y axis). Clusters are spread on the x axis, with the probability of expression of the markers figured by the size of the black dots.

Extended Data Fig. 4 Marker gene expressions in TmY4, TmY8, TmY14, LC12 and LC17 neurons.

a, Expression pattern of TmY4-LexA (green) in the adult optic lobe (n = 7 brains) with anti-Ncad immunostaining (white). The LexA line shows weak or no expression in the most anterior medulla region. bd, Expression pattern of TmY4-LexA (green) in the adult optic lobe with anti-Dichaete (D) (b), anti-Vvl (c) and anti-Toy (d) immunostainings (magenta), n = 15 neurons for each. TmY4 cell bodies express Dichaete but not Vvl or Toy. e, Sparse labelling of cell types (anti-Flag, green) expressing CG42548 in the adult optic lobe using MCFO and immunostained with anti-Brp (white) and anti-Vvl (magenta). A TmY8 neuron (n = 4 neurons) is labelled (arborized layers shown by arrows) and expresses Vvl. Adult flies were heat-shocked for 5 min and dissected after 6 days. fh, Expression pattern of R24F10-Gal4 (green) in late L3 optic lobes with anti-Ncad (white, f), anti-Dac (magenta, f), anti-Dichaete (magenta, g) and anti-Toy (magenta, h) immunostainings, n = 15 neurons for each. R24F10-Gal4 is expressed in TmY14 neurons during larval stage (unpublished data). TmY14 neurons express Toy (h) but not Dac or D (f, g). i, Expression pattern of LC12-Gal4 (green) in the adult optic lobe (n = 7 brains) with anti-Ncad immunostaining (magenta) showing processes in lobula layers 2–4, and a projection to an optic glomerulus in the posterior ventrolateral protocerebrum (PVLP) of the central brain (white outline). The cell bodies of LC12 are located in the lateral cell body rind, the region separating the optic lobe and central brain. j, Expression pattern of beat-Ic (green) in the adult optic lobe (n = 6 brains) with anti-Ncad immunostaining (magenta) showing a strong expression profile in the optic glomerulus corresponding to LC12 in the PVLP (white outline). km, Expression pattern of LC12-Gal4 (green) in the adult optic lobes with anti-Cut (k, magenta-top panel, grey-bottom panel), anti-Acj6 (l, magenta, top panel; grey, bottom panel), and anti-Toy (m, magenta, top panel; grey, bottom panel) immunostainings, n = 77 neurons. All LC12 cells bodies are cut+, Acj6+ and toy+. Red line delineates LC12 cell body location. n, Expression pattern of LC17-Gal4 (green) in the adult optic lobe (n = 5 brains) with anti-Ncad immunostaining (magenta) showing processes in lobula layers 2–5. LC17 project to the PVLP optic glomerulus (white outline) adjacent to that of LC12 (asterisk, see also i). o, Expression pattern of Kn-Gal4 (green) in the adult optic lobe (n = 3 brains) with anti-Ncad immunostaining (magenta) showing a strong expression profile in the optic glomerulus corresponding to LC17 in the PVLP (white outline). Note its absence from LC12 neurons (asterisk). p, q, Expression pattern of LC17-Gal4 (green) in the adult optic lobes with anti-Acj6 (p, magenta, top panel; grey, bottom panel) and anti-Toy (q, magenta, top panel; grey, bottom panel) immunostainings, n = 62 neurons. All LC17 cells bodies are Acj6+ and toy+. Red line delineates LC17 cell body location (p, q). Me, medulla; Lo, lobula; LoP, lobula plate. Scale bars: 25 μm (aj, n, o), 15 μm (km, p, q).

Extended Data Fig. 5 Overview of the final adult clusters.

a, Hierarchical clustering tree of our adult clusters, based on the average log-normalized non-integrated expression of the 2,000 most variable genes of the dataset (the genes used to define the unsupervised clusters, Methods). Indicated in blue are the glial clusters. b, Number of cells in our identified neuronal clusters, excluding the T4–T5 cluster that contains 10,780 cells, with unicolumnar neurons in blue and multicolumnar neurons in orange. Importantly, photoreceptors (PR) and lamina neurons L1–L5 clusters contain fewer cells, as these neuronal types were not included in equal proportions in all libraries. Because the number of cells per optic lobe for a given neuronal type is rarely formally counted, unicolumnar versus multicolumnar character is based both on general knowledge and the following references7,8,12,55.

Extended Data Fig. 6 Benchmarking of the neural network classifier.

a, b, t-SNE visualization of the P70 optic lobe single-cell transcriptomes, using 120 principal components calculated on the log-normalized integrated gene expression. Cells colours indicate the clusters they belonged to according to unsupervised clustering (a), or the adult clusters they were classified as by the neural network (b, same as in Fig. 1e). Black circles indicate high granularity regions, where less frequent cell types were grouped together by unsupervised clustering but could be resolved accurately by the neural network. c, Same as in a, b but cells are named and coloured by the adult cluster they were classified as by Seurat label transfer (Methods). d, t-SNE visualization (same as c) including only the cells that were assigned inconsistent identities by Seurat and the neural network. Highest rates of inconsistencies were observed in the centre (LQ cells), in L1 and L2 clusters (red ellipses), in most glia clusters (green ellipses), the TE neurons and a glia-like cluster (identity 214, Supplementary Table 1) with no adult correspondence (blue ellipses). e, f, t-SNE visualizations of 56,902 cells sequenced from whole fly brains19, using 120 principal components calculated on the log-normalized gene expression. e, Cells are named and coloured by the clusters they were classified as by our neural network. f, Cells are named by the cluster identities from the original study and coloured by the confidence score they received from our neural network. Black circles mark the following central brain clusters (from left to right): Poxn, OPN, clock neurons and dopaminergic neurons, that all received low scores from the neural network. Kenyon cells (red circles) were assigned with high confidence as our adult dataset was contaminated by them (cluster 112).

Extended Data Fig. 7 High-resolution transcriptomic atlases of the optic lobe across development.

af, t-SNE visualizations of all optic lobe single-cell transcriptomes acquired for this study, using 120 principal components calculated on the log-normalized integrated gene expression. The cells are named and coloured consistently at all stages by the neural network classifications with manual adjustments as detailed in Methods. Blue ellipses highlight Dm3 and Tm9 neuronal subtypes, which could only be resolved at P50 and earlier.

Extended Data Fig. 8 Transient extrinsic neurons.

a, b, t-SNE visualization of the P70 optic lobe single-cell transcriptomes, using 120 principal components calculated on the log-normalized integrated gene expression. Cells are named by the unsupervised cluster they were assigned to and coloured by the confidence score they received from the neural network (NN) (a) or by the log-normalized non-integrated expression of Fs (green), dimm (blue), and skl (red) (b), which are co-expressed in TE neurons (red ellipses). c, Violin plot of log-normalized non-integrated prt expression in all clusters at P50. TE neuron clusters are indicated by the circle. d, R10D10-Gal4 co-expression with anti-Prt staining in a P50 optic lobe (n = 15 neurons). Scale bar, 10 μm. e, FLEXAMP memory cassette labelling of R10D10-Gal4 in an adult optic lobe (n = 28 brains) with anti-Ncad staining. Scale bar, 30 μm. f, R10D10-Gal4 expression pattern in L3 optic lobe (n = 15 brains), with anti-Ncad, anti-Bsh and anti-Hth staining. Arrow indicates Bsh+Hth neurons labelled by R10D10-Gal4. Scale bar, 30 μm. g, h, R10D10-Gal4 sparse expression at P30 (n = 40 neurons), with anti-Ncad, anti-Bsh and anti-Hth staining. Scale bars, 5 μm (g) and 15 μm (h). dMe, distal Medulla; pMe, proximal Medulla; Lo, Lobula; Lp, Lobula plate. i, Co-labelling of R10D10-LexA expression and bsh-Gal4 FLEXAMP memory cassette with anti-Ncad staining in a P50 optic lobe (n = 13 brains). Dashed ellipses highlight TE neurons. Scale bar, 20 μm.

Extended Data Fig. 9 Early differentiation and transcriptomic synchronization of optic lobe neurons and summary of the main findings.

a, b, t-SNE visualization of the P15 optic lobe single-cell transcriptomes, using 120 principal components calculated on the log-normalized integrated gene expression. Cells are named by the unsupervised cluster they were assigned to and coloured by the confidence score they received from the neural network (a) or by the log-normalized non-integrated expression of dpn (green), ase (blue), and grim (red) (b). Circles match those of Fig. 3a. c, UMAP visualization of the P15 optic lobe single-cell transcriptomes, using 120 principal components calculated on the log-normalized integrated gene expression. Cells are coloured by the log-normalized non-integrated expression of nerfin-1 (green), Hey (blue), and vfl (red). d, UMAP visualization of Tm3 and T1 cells (above and below the dashed line, respectively) from all stages sequenced in this study, using 25 principal components calculated on the log-normalized non-integrated gene expression. Cells are coloured by their developmental stage. e, Ventral and dorsal transient extrinsic (TE) neurons as well as transient photoreceptors (PRs) line the edges of all optic lobe neuropils and express Follistatin (Fs). Moreover, TE and at least three other neuronal types express Wnt4 in the ventral medulla/lobula but express Wnt10 in the dorsal part of these neuropils. f, The transcriptome of neurons from the same neuronal type but produced days apart converge towards a similar transcriptomic state, which they reach by P30. Moreover, the inter-neuronal type transcriptomic diversity is highest during P40–P70.

Extended Data Fig. 10 Increased transcriptomic diversity during synaptogenesis.

a, Log-normalized and scaled (to the max of 1 for each gene across all time points) expression of top10 (by logFC, calculated at P50) subcluster markers between T4 and T5 subtypes at all stages. TF, transcription factor; CSM, cell-surface molecule. b, c, GO enrichment analysis of all (269) differentially expressed genes between the T4–T5 subclusters at P50. All Biological Process (b) and Molecular Function (c) terms with greater than twofold enrichment were summarized by REVIGO to eliminate redundant terms and group related ones together. Inner rings in the CirGO plots indicate the summarized terms (names in bold). Some individual terms (outer ring) relevant to neuronal function and development are also labelled. Areas within the graphs are determined by the P values of the terms. d, Average normalized number of presynaptic sites from Dm3 neurons with orthogonally oriented dendritic arbors (because we could not assign the anatomical directions from the EM data, we plotted them as Dm3x and Dm3y) to indicated neuronal types (Methods). Only the outputs showing differences between the subtypes are plotted. Error bars denote s.e.m. P = 0.004 (Dm3x), 0.006 (Dm3y), 0.04 (Tm28), unpaired parametric two-tailed t-tests (n = 5 and 7 Dm3 cells per type). e, Number of genes differentially expressed between the indicated subtypes with a P < 0.01 (two-sided Wilcoxon rank-sum test), calculated after downsampling the number of cells in each group to 150 for consistency.

Extended Data Fig. 11 GO enrichment analysis of the cluster and stage markers.

a, b, GO terms were determined from the most differentially expressed genes between neuronal clusters (ranked top20 by logFC for at least one cluster) at all stages separately (a) or all genes differentially expressed between stages (Methods) in all neurons (aggregated) (b). All terms with greater than twofold enrichment were summarized by REVIGO to eliminate redundant terms and group related ones together. Inner rings (labelled within the graphs or in bold) indicate the summarized terms. Some individual terms (outer ring, not bold), if non-redundant with the summary terms, are also labelled. Areas within the graphs are determined by the P values of the terms.

Extended Data Fig. 12 Dorsal and ventral visual circuits are partitioned by differential Wnt signalling.

a, Tm9-Gal4 sparse labelling in the ventral and dorsal part of the same adult optic lobe (n = 4 neurons) with anti-Ncad immunostaining (grey). b, Expression pattern of Wnt4-Gal4 (green) with anti-Chaoptin immunostaining (magenta) in the adult optic lobe (n = 8 brains). c, Expression pattern of Wnt4-Gal4 (green) with anti-Chaoptin (magenta) and anti-Dve (cyan) immunostainings, in the ventral retina at P30 (n = 6 eye discs). Chaoptin marks all photoreceptors, and Dve is expressed in the photoreceptors R1-6 and a subset of R7 (yellow R7), but not in R8 and the rest of R756. d, Expression pattern of Tm4-Gal4 (green) with anti-Aop immunostaining (magenta) at P50 (n = 3 brains), showing that almost all Aop+ neurons are co-labelled by Tm4-Gal4. Arrowheads show Aop+ neurons that are not co-labelled by Tm4-Gal4. e, Wnt4-Gal4 expression pattern with anti-Aop staining (Tm4 marker, see d) at P50 (n = 6 brains). Aop+ neurons co-express Wnt4-Gal4 in the ventral (white arrows) but not dorsal (white arrowheads) optic lobe. f, fz2, mamo or CG9896 differential expression between either Tm9v and Tm9d cells, TEv and TEd cells, Wnt4+Wnt10 and Wnt4Wnt10+ Tm4 cells or Wnt4+Wnt10 and Wnt4Wnt10+ cluster 62 cells (log-normalized non-integrated expression). Two-sided Wilcoxon rank-sum test, P values are indicated on the figure, NS, not significant. Scale bars, 10 μm (a, c) or 30 μm (b, d, e).

Supplementary information

Supplementary Information

This file contains the Supplementary Discussion and Supplementary References.

Reporting Summary

41586_2020_2879_MOESM3_ESM.pdf

Supplementary Figure 1 FACS Gating Strategy. This figure contains plots exemplifying the gating strategies used for sorting specific cell types for the bulk sequencing experiments.

41586_2020_2879_MOESM4_ESM.xlsx

Supplementary Table 1 Details on the annotation of the adult and pupal datasets. This table indicates how the annotation of our adult clusters was determined, gives details about how these annotations were transferred to pupal clusters (i.e. if cells were discarded in the process, if new identities were created, etc.), and list general observations we made about the clusters but did not list in main text (i.e. if a cluster could include low quality transcriptomes, potential identifications for some clusters based with their proximity with other cell types, etc.).

41586_2020_2879_MOESM5_ESM.xlsx

Supplementary Table 2 Unique transcription factor combinations in the neuronal clusters. Please refer to the Methods subsection named “Stable transcription factor markers in neuronal clusters” for details.

Supplementary Table 3 Origins of fly strains and antibodies used.

41586_2020_2879_MOESM7_ESM.zip

Supplementary Data Appendix 1. This zipped file includes the instructions, Python/R code and example datasets to locally run the neural network classifier.

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Özel, M.N., Simon, F., Jafari, S. et al. Neuronal diversity and convergence in a visual system developmental atlas. Nature 589, 88–95 (2021). https://doi.org/10.1038/s41586-020-2879-3

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