Fig 1.
CB and VNC neural lineages identified by scRNA-Seq in wandering larvae brain.
(A) VT201094-Gal4 driving expression of UAS::CD8-GFP. Expression occurs in neural lineages from CB and VNC, but not the OL. Views from anterior and posterior sides; Dpn (magenta), GFP (green); scale bar, 50 μm. (B) Close-up of one type I neural lineage, outlined, in the anterior side of CB; Dpn (white), Ase (magenta), GFP (green), Elav (blue); scale bar, 10 μm. (C) Schematic representation of type I neural lineages; cells colored by expression of markers as described; green outline indicates GFP expression and thus the cells in which VT201094-Gal4 drives expression in an 18-h time window; frequency of cell division is indicated in hours. (D) UMAP visualization of scRNA-Seq dataset composed of 12.7K cells of neural lineages from CB and VNC; clusters colored based on cell type annotation; dashed arrow indicates the direction of the neural differentiation trajectory. (E) Dot plot showing genes that were used to identify each cell type. Dot size indicates the percentage of cells expressing the gene in each cell type; color variation represents the average expression of the gene in each cell type. The data underlying this figure are contained within GEO database (accession number: GSE179763); see also S1 and S2 Figs. CB, central brain; dINP, developing INP; GMC, ganglion mother cell; INP, intermediate neural progenitor; NB, neuroblast; OL, optic lobe; scRNA-Seq, single-cell RNA sequencing; VNC, ventral nerve cord.
Fig 2.
Transcriptomic differences in neurons identify 2 phases of maturation.
(A) Feature plot for the marker of young neurons Hey. (B) Feature plots for the neuron maturation markers nrv3 (right) and nSyb (left). (C) Chord diagrams showing the correspondence between ion channels (left) and immunoglobulin and cadherin super families (right) and the clusters in which these genes appear as top markers (top 5 markers). (D) Feature plots for neurotransmitter pathway identity markers (VGlut, ChAT, VAChT, Gad1, Vmat); these markers are predominantly expressed in older neurons. (E) Representative illustration of the proposed neuronal maturation phases in a UMAP plot: Phase 1 (younger/less mature) and Phase 2 (older/more mature). (F) Heatmap with the top 100 genes most differentially expressed throughout pseudotime in neurons; differential expression analysis performed with monocle. The data underlying this figure are contained within GEO database (accession number: GSE179763); see also S3 Fig and S1 and S2 Tables. ChAT, choline acetyltransferase; Gad1, glutamic acid decarboxylase 1; nrv3, nervana 3; nSyb, neuronal Synaptobrevin; UMAP, uniform manifold approximation and projection; VAChT, Vesicular acetylcholine transporter; VGlut, vesicular glutamate transporter; Vmat, Vesicular monoamine transporter.
Fig 3.
The timing of VGlut and ChAT protein detection depends on animal age.
(A, B) 18-h clones induced at 87 h ALH with the VT201094-Gal4 driver using G-TRACE; VT201094-Gal4;tubGal80ts was used to permanently label with GFP NB-derived lineages using G-TRACE; clones were analysed at 105 h ALH (wandering third instar larvae); oldest neurons in the clone are approximately 12.5 h old. (A) smFISH against VGlut or ChAT. Individual mRNA molecules are displayed as magenta dots; neural cells are labeled with nuclear GFP (green). Dashed line delimitates a neural clone; pink arrowhead indicates the closest cell to the neuroblast where mRNA is visible; yellow asterisk identifies neuroblast. Z-projection from 21 slices with 0.25 μm interval, resulting in a total z range of approximately 5 μm (roughly the size of a neuron); scale bar, 5 μm. (B) Immunofluorescence images for VGlut or ChAT antibody staining (magenta). Neural cells in clones are labeled with nuclear GFP (green); outlines indicate examples of lineages with GFP-positive cells; yellow asterisk identifies neuroblast. Scale bar, 20 μm. (C, D) 72-h clones induced at 87 h ALH and analysed at 159 h ALH (approximately 48 h APF) using G-TRACE; oldest neurons in the clone are approximately 66.5h. (C) Schematic representation of the temporal strategy used to induce 72-h clones in neural lineages represented in green. Timings used for the scRNA-Seq dataset (18-h clones induced at 87 h ALH and analysed at 105 h ALH) are represented in grey. (D) Immunofluorescence images for Vglut or ChAT antibody staining (magenta). VT201094-Gal4 drives nuclear GFP (green) expression in NBs, daughter cells generated by NBs during clone period inherit GFP expression. Outlines indicate clones; scale bar, 20 μm. (E, F) 72-h clones induced at 50 h ALH and analysed at 122 h ALH (approximately 12 h APF) using G-TRACE; oldest neurons in the clone are approximately 66.5 h. (E) Schematic representation of temporal strategy used to induce clones in neural lineages. (F) Immunofluorescence images for VGlut or ChAT antibody staining (magenta). VT201094-Gal4 was used to drive nuclear GFP (green) expression in NBs; outlines indicate neurons clones; scale bar, 20 μm. See also S4 Fig. ALH, after larval hatching; APF, after puparium formation; ChAT, choline acetyltransferase; NB, neuroblast; scRNA-Seq, single-cell RNA sequencing; smFISH, single-molecule fluorescence in situ hybridization; VGlut, vesicular glutamate transporter.
Fig 4.
VGlut and ChAT proteins are not detected in wL3 OL.
(A) insc-Gal4,UAS-CD8::GFP was used to label NB lineages in the OL. GFP(cyan), VGlut (yellow), and ChAT (magenta) protein expression in the OL at wL3 stages, 0 h APF, and 24 h APF. Insc-Gal4 is expressed in both OL and CB regions; OL is delimitated by a dashed line. Arrows in 24 h APF panel refer to VGlut protein (yellow) or ChAT protein (magenta) expression. Scale bar, 50 μm. (B, C) Mean fluorescence intensity measured in wL3 raised in food supplemented with MG132 diluted in DMSO, or in DMSO alone (control). (B) Arm mean fluorescence intensity was measured in OL neuroepithelia (control, n = 6; MG132, n = 5). (C) VGlut and ChAT mean fluorescence intensity was measured in medulla neurons (control, n = 7; MG132, n = 5). Plotted data were normalized to the mean of the control. Data shown as mean ± SEM; statistical analysis was done using unpaired two-tailed t test; **P value < 0.01, ns = nonsignificant. The data underlying this figure are contained within S3 Data. (D) UAS-Stinger::GFP (green) expression driven by VGlut-T2A-Gal4 or ChAT-T2A-Gal4 in wL3 stages. DAPI (magenta). OL delimited by dashed line. CB located to the left of the OL. Scale bar, 50 μm. (F) Model for the asynchronous onset of VGlut and ChAT transcription and translation during the first phases of neuron maturation. See also S4 Fig. APF, after puparium formation; Arm, Armadillo; CB, central brain; ChAT, choline acetyltransferase; NB, neuroblast; OL, optic lobe; VGlut, vesicular glutamate transporter; wL3, wandering L3.
Table 1.
Resources table.