Cell atlas of central brain with single nuclei RNAseq.

(A-A’) Genetics (A) to label progeny (A’) derived from T2 NBs. Dashed lines show the boundary of optic lobes and central brain, and the optic lobes were removed during dissection.

(B) Central brain atlas labeled with known cell types. Abbreviations: CLK, clock neurons; CRZ, Corazonergic neurons; DOP, dopaminergic neurons; HEM, hemocytes; MBN, mushroom body neurons; OCTY, octopaminergic-tyraminergic neurons; OL, optic lobe; OPN, olfactory projection neurons; SER: serotoninergic neurons.

(C) Central brain atlas labeled by NB (T1 or T2) lineage. Dash line-outlined box shows the region enriched with the cells derived from T2 NBs, and the identity are shown at the bottom-right box.

(D) Dot plot of top 5 marker genes of the T2-enriched clusters.

(E) Atlas of central brain glia labeled with known cell types. (E’) T1 and T2 derived cells colored cyan and red respectfully.

(F) Dot plot of known and top marker genes of glial clusters.

Cell atlas of neurons produced by T1 or T2 NBs.

(A) Cell atlas from T2 NBs. (B) Heatmap of scaled average expression of top 10 markers genes from each T2 neuronal cluster.

Expression of fast-acting neurotransmitters in neurons derived from T2NSC lineages.

(A-G) UMAP distribution plots demonstrate the expression of the following neurotransmitters: (A) vesicular glutamate transporter(VGlut, glutamatergic neurons), (B) vesicular acetylcholine transporter(VAChT, cholinergic neurons), (C) glutamic acid decarboxylase 1(Gad1, GABAergic neurons), (D) tyramine β hydroxylase (Tbh, tyraminergic neurons), (E) tyrosine 3-monooxygenase(Ple, dopaminergic neurons), (F) serotonin transporter(SerT, serotonergic neurons) (G) tyrosine decarboxylase (Tdc2, octopaminergic neurons). All plots have a minimum cutoff value set at 0.

(H) The UpSet quantifies the number of cells in the atlas that express each neurotransmitter gene with a scaled expression > 2 (Gu, 2022).

Glial cell types present in the T2-derived cell atlas.

(A) Sub-clustered of 12,315 nuclei from Repo+ T2 clusters in UMAP distribution.

(B) Dot plot of validated glial subtype markers to identify glial clusters by differential gene expression.

Sex differences present in the T2-derived glia and neurons.

(A-A’’) T2glia in UMAP distribution across samples: (A) T2 Female (3,910 nuclei), (A’) T2 Male (4,385 nuclei), (A’’) Female and Male mixed (4,020 nuclei).

(B) Biased clusters for male to female ratio for number of nuclei in the T2 glia clusters.

(C) Differential expression between male and female T2 glia.

(D) Heatmap of top differential gene expression between male and female nuclei within glial clusters.

(E-E’’) T2neuron in UMAP distribution across samples: (E) T2 Female (8,151 nuclei), (E’) T2 Male (16,201 nuclei), (E’’) Female and Male mixed (25,796 nuclei).

(F) Biased clusters for male to female ratio for number of nuclei in the T2 neuron clusters.

(G) Differential expression between male and female T2 neurons.

(H) Heatmap of top differential gene expression between male and female nuclei within neuron clusters.

Neuropeptide expression in T2-derived neurons.

(A) Dot plot showing expression of 13 neuropeptides and their co-expression with the 7 fast-acting neurotransmitters (red dashed line) across selected clusters.

(B) Genetic scheme to map neuropeptide expression in T2-derived adult neurons.

(C-E) Neuropeptide-expressing neurons labelled with GFP in three-dimensional projections. (C’-E’) Fan-shaped body projections. (C’’-E’’) Ellipsoid body projections. nc82 counterstains (magenta) in the brain for neuropil projections.

(F) Heatmap showing the top 5 transcription factors most strongly correlated with each neuropeptide across all cells (Sigorelli, 2024). Scale bar represents 20 μm.

Combinations of transcription factor expression in T2-derived neurons.

Binarized heatmaps of positive (A) zinc finger (B) helix-turn-helix (C) homeodomain TF markers in T2-derived neurons. Clusters are sorted by similarity based on Jaccard index scores.

(C’) Top five marker genes for clusters which had non-unique combinations of homeodomain TF expression.

(D) Percentage of clusters with unique combinations of TF expression based on TF class (zinc finger TFs = 100% (161 unique clusters), helix-turn-helix TFs = 100% (161 unique clusters), homeodomain TFs = 93.8% (150 unique clusters), basic domain TFs = 69.6% (112 unique clusters), unidentified DNA binding domain TFs = 59% (95 unique clusters), high motility group TFs = 34.2% (55 unique clusters).

Mapping T2-derived neurons to UMAP clusters.

(A) Heatmap of the coefficients (r) of expression profiles of single cell clusters (columns) to the expressions of known central complex neurons (rows) profiled with bulk RNA sequencing. The matching of the cluster to the central complex neurons is determined by the highest coefficient value (gray box) for each cluster. FB6A(1) and FB6A(2) are labeled and profiled with two different split-gal4 drivers (Wolff et al., 2024). Cluster 75 and 95 identified as E-PG were verified in Dillon et al., 2024.

(B) Scaled average expression of the enhancer genes of split-gal4 drivers (columns) in each cluster (rows). The name of central complex neurons (CX) labeled by split-gal4 drivers are listed at the top. The cluster identity was determined by the combination of positive scaled average expression of the enhancer genes (black boxes).

(C) Dotplot of the known marker genes (toy, runt) expressed in central complex neurons (PF-R, P-FN, and P-EN), lexA-driver enhancer gene (rho, Gγ30A), split-gal4-driver (SS00191) enhancer genes (shakB, Pkc53E), and newly identified marker genes.

(D) PF-R (R37G12-Gal4 UAS-V5) neuronal cell bodies labeled with V5, and co-stained with marker genes boxed in (C).

(E,F) PF-N (R16D01-Gal4 UAS-V5) neuronal cell bodies labeled with V5, and co-stained with marker genes boxed in (C).

(G) PE-N (R12D09-Gal4 UAS-V5) neuronal cell bodies labeled with V5, and co-stained with marker genes boxed in (C).

(H) Percentage of marker genes in PE-N and PF-N. Scale bar: 5 μm in all panels.

Summary.

Asterisk, known neuron-cluster associations; others are predicted neuron-cluster associations. Underline, neurotransmitter expressing clusters. Abbreviations: AstC, Allatostatin C+ neurons; CCAP, Crustacean cardioactive peptide+ neurons; DOPA, dopaminergic neurons; dsx, doublesex+ neurons; MBN, mushroom body neurons; OCTY, octopaminergic-tyraminergic neurons; Proc, Proctolin+ neurons; Tk, Tachykinin+ neurons; SER, serotonergic neurons. E-PG, FB1C, FB2A, FB2B, FB2I_ab, FB3C, FB4K, FB4L, FB6A, FB6H, FB7B, FB8B, FB8G, FR1, hΔA, hΔD, hΔE, hΔH, hΔI, hΔK, lbSps-P, P-EG, P-EN, P-FGs, P-FN, PF-R, vΔC and vΔE are the names of central complex neurons (Hulse et al., 2021).