Overview on the Tc-foxQ2II positive cells.

Neuronal projection patterns were identified using immunohistochemistry against EGFP in the adult brain of foxQ2-5’-line. (A-D) Different parts of the adult brain stained for EGFP (green), Synapsin (magenta), and DAPI (blue). (A’-D’) The EGFP channel is shown with the brain outline indicated by dashed lines. (A-A’) Overview of a projection of the entire brain; (B-B’) anterior subsection; (C-C’) medial subsection; (D-D’) posterior subsection. White arrowhead: Lateral Cluster 1; white arrow: Lateral Cluster 2; open arrowhead: Lateral Cluster 3; open arrow: Lateral Cluster 4; yellow arrowhead: Median Cluster 1; yellow arrow: Median Cluster 2; yellow dashed circle: Posterior Cluster; white dashed circle: Tritocerebrum Cluster; white star: Anterior Cluster. Scale bar: 100 μm.

Overview on the reconstruction of the nine Tc-foxQ2II positive cell clusters.

(A) 3D reconstruction of major neuropils and EGFP-positive clusters of neurons based the brain shown in Fig. 1. Posterior view in (A) and anterior view in (A’). The cluster AC (white star in A’) was not analyzed further as it was not Tc-foxQ2II positive. White circle: Tritocerebral cluster; yellow circle: Posterior cluster 1 and 2. (B) Schematic overview of the cell body locations for the ten clusters shown in the same colors as in A/A’ and subsequent figures. White arrowhead: Lateral Cluster 1 (orange); white arrow: Lateral Cluster 2 (yellow); open arrowhead: Lateral Cluster 3 (purple); open arrow: Lateral Cluster 4 (dark purple); yellow arrowhead: Median Cluster 1 (magenta); yellow arrow: Median Cluster 2 (pink); yellow dashed circle: Posterior Cluster (PC1 is olive green and PC2 is vibrant green); white dashed circle: Tritocerebrum Cluster (light green and white broken circle in A); white star: Anterior Cluster (grass yellow). Scale bar: 100 μm.

The morphology and distribution of EGFP-positive clusters.

(A-A’) Posterior view and anterior views of the brain, showing major neuropils. (B-B’) Median cluster 1 (MC1). (B) Posterior view. (B’) Lateral view. The region enclosed by the red dashed square is magnified in the bottom-left corner, with a white arrow indicating the specific projection area. (C-C’) Median cluster 2 (MC2). (C) Posterior view. (C’) Lateral view. The central complex (CX) region, enclosed by a red dashed square, is magnified in the top-right corner. A white arrow indicates projections targeting the VMCpo/PS and LAL. (D-D’) Lateral cluster 1 (LC1). (D) Posterior view. (D’) Anterior view. (E-E’) Posterior view of lateral cluster 2 (LC2). In (E’), a white arrow marks the VMCpo/PS region traversed by LC2. Abbreviations: AL: Antennal lobe; CB: Central body (CBU: Upper unit of the central body; CBL: Lower unit of the central body); LH: Lateral horn; MB: Mushroom body (CA: Calyx; ML: Medial lobe; PED: Peduncle; VL: Vertical lobe); OL: Optic lobe; PB: Protocerebral bridge; SLP: Superior lateral protocerebrum; SMP: Superior medial protocerebrum; TR: Tritocerebrum; VMCpo/PS: Ventromedial cerebrum postcommissural domain / Posterior slope.

The morphology and distribution of EGFP-positive clusters in the brain of the foxQ2-5’ line

(A- A’) Lateral cluster 3 (LC3). (A) Posterior view. (A’) Anterior view. (B-B’) Lateral cluster 4 (LC4). (B) Posterior view. (B’) Lateral view. A white arrow indicates LC4 arborization in the LH and SLP. (C-C’) Posterior cluster 1 (PC1). (C) Posterior view. (C’) Lateral view. The region enclosed by the red dashed square is magnified in the top-right corner. A white arrow shows where PC1 enters the VMCpo/PS. (D-D’) Posterior cluster 2 (PC2). (D) Posterior view. (D’) Lateral view. The projection region, enclosed by a red dashed square, is magnified in the top-right corner. A white arrow points to the arborization site. (E) Anterior view of tritocerebrum cluster (TC). A white arrow indicates its arborization zone. Abbreviations: AL: Antennal lobe; CB: Central body (CBU: Upper unit of the central body; CBL: Lower unit of the central body); LH: Lateral horn; MB: Mushroom body (CA: Calyx; ML: Medial lobe; PED: Peduncle; VL: Vertical lobe); OL: Optic lobe; PB: Protocerebral bridge; SLP: Superior lateral protocerebrum; SMP: Superior medial protocerebrum; TR: Tritocerebrum; VMCpo/PS: Ventromedial cerebrum postcommissural domain / Posterior slope.

Classification of Lateral Cluster 1 based on distinct projection patterns.

(A-A’) Morphology and projection pattern of LC1a, shown without (A) and with (A’) brain compartments. The region enclosed by the red dashed square shows where LC1a passes near the central body (CB) before crossing the midline. A magnified view of this region (top-right corner) shows the LC1a projection adjacent to the CB, indicated by a white arrow. (B-B’) LC1b projections. The boxed region in (B’) shows that LC1b projects toward the CB. A magnified lateral view (top-right) details the contact point (white arrow). Abbreviations: VL: the vertical lobe; ML: the medial lobe; CBU: the upper unit of the central body.

Eight clusters detected in the foxQ2-5’– line were also sparsely labelled by Brainbow.

(A) MC1. (B) MC2. (C) LC1. (D) LC2. (E) LC4. (F) PC1. (G) PC2. (H) TC. All panels show the brain of the foxQ2-5’- line. Magenta represents synapsin. Scale bar: 100 μm.

EGFP-positive cells arborize in the mushroom body and the central complex.

(A-A’’) Arborizations of EGFP-positive cells in the mushroom body (MB). (A) Overview of 3D reconstruction of the MB and EGFP-positive projections, generated using VVDviewer. (A’) Hemi-mushroom body with EGFP-positive projections. (A”) Immunohistochemistry result showing the MB and EGFP-positive projections. White arrows in (A’) and (A’’) indicate EGFP-positive projections arborizing in the α and α’ lobe of the MB. (B-B’’) Arborizations of EGFP-positive cells in the central body (CB). (B) Overview of the 3D reconstruction of the CB with EGFP-positive projections. (B’) Side profile view of the area marked by the white dashed rectangle in (B). (B’’) Immunohistochemistry result showing the CB and EGFP-positive projections. White arrowheads in (B’) and (B’’) indicate EGFP-positive projections arborizing in the upper unit of the central body (CBU). Open arrowheads in (A’) and (B’’) indicate the EGFP-positive projections arborizing in the peduncle (PED). The White dashed circle in (B’’) outlines the PED. Green, EGFP; magenta, synapsin; and white, neuropils (MB or CB).

Reconstruction of STED-imaging of LC1a connection of mushroom body with the central complex in the foxQ2-5’- line brain.

(A) LC1a seems to have two main branches: one innervates the mushroom body (MB), and the other innervates the upper unit of the central body (CBU). (B) LC1b arborizes only within the central body (CB). (C) A higher-magnification view of the projections in the CBU (region partially outlined by the white dashed rectangle in A). (D-F) Transverse sections through distinct domains of the central body. (D) Median section. (E) A lateral section showing a single branch projecting into the CB. (F) A section where two distinct tracts are visible. Green and magenta represent the main projections from different sides, which are in close proximity around the midline (white). Yellow indicates arborizations within the CBU. CBU is shown in grey. CBL: lower unit of the central body. The STED dataset used in this panel was acquired by Ying.

Neurotransmitter expression in different EGFP-positive cell types in the foxQ2-5’-line brain

Grey indicates clusters that contained at least one neuron expressing the respective transmitter. Hence, it does not mean that all cells of a cluster have a respective transmitter nor that transmitters are co-expressed in the neurons.

A subset of Tc-FoxQ2-positive neurons are dopaminergic.

Co-immunostaining for GFP (green) and the dopamine marker Tyrosine Hydroxylase (TH, magenta) identifies dopaminergic subtypes among the Tc-FoxQ2-positive neuronal populations. Dashed outlines indicate the brain contour. (A-A’’) The anterior brain region containing lateral cluster 3 (LC3, white arrowheads). (A’) GFP channel. (A’’) TH (dopamine) channel. (B-B’’) A medial section showing the median cluster (MC, open arrowheads), and lateral cluster 2 (LC2, white arrows). (C-C’’) A posterior section showing the median cluster (MC, open arrowheads), lateral cluster 1 (LC1, open arrows) and lateral cluster 2 (LC2, white arrows). Scale bar: 100 μm.

Co-expression of Tc-foxQ2II and TH (dopamine) in distinct neuronal subsets.

A magnified view highlights neuronal subtypes co-expressing the dopaminergic marker Tyrosine Hydroxylase (TH) and Tc-FoxQ2/EGFP, alongside an analysis of the signal overlap ratio. (A-D’’) A representative imaging plane showing co-immunostaining for neurons expressing both EGFP (green) and TH (magenta). (E) Schematic of the left-brain hemisphere. Four Tc-FoxQ2-positive neuronal subtypes are outlined with differently colored dashed circles. (F) Venn diagrams quantify the overlap between TH-positive and Tc-FoxQ2-positive neurons for each of the four subtypes (n = 5). Scale bar: 20 μm.

3D reconstruction of Tc-foxQ2-Dopaminergic neuronal projections in the brain of foxQ2-5’-line using VVDviewer.

(A) Overview of the projections from the four neuronal subtypes. (B) Projections from the median cluster (MC). MC1 contains two long tracts (indicated by a white arrowhead), consistent with previous descriptions. (C) Projections from all subtypes within lateral cluster 1 (LC1). (D-D’’’) Individual projections of each LC1 subtype. (D) Overview of the hemi-LC1. (D’) LC1a projection (see also Fig. 5). (D’’-D’’’) LC1b projections (see also Fig. 5). (E) Projections from LC2 and LC3.