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. 2023 Nov 29;18(1):9.
doi: 10.1186/s13064-023-00177-9.

Imp is expressed in INPs and newborn neurons where it regulates neuropil targeting in the central complex

Jordan A Munroe  1 , Chris Q Doe  2
Affiliations

Imp is expressed in INPs and newborn neurons where it regulates neuropil targeting in the central complex

Jordan A Munroe et al. Neural Dev. .

Abstract

The generation of neuronal diversity remains incompletely understood. In Drosophila, the central brain is populated by neural stem cells derived from progenitors called neuroblasts (NBs). There are two types of NBs, type 1 and 2. T1NBs have a relatively simple lineage, whereas T2NBs expand and diversify the neural population with the generation of intermediate neural progenitors (INPs), contributing many neurons to the adult central complex, a brain region essential for navigation. However, it is not fully understood how neural diversity is created in T2NB and INP lineages. Imp, an RNA-binding protein, is expressed in T2NBs in a high-to-low temporal gradient, while the RNA-binding protein Syncrip forms an opposing gradient. It remains unknown if Imp expression is carried into INPs; whether it forms a gradient similar to NBs; and whether INP expression of Imp is required for generating neuronal identity or morphology. Here, we show that Imp/Syp are both present in INPs, but not always in opposing gradients. We find that newborn INPs adopt their Imp/Syp levels from their parental T2NBs; that Imp and Syp are expressed in stage-specific high-to-low gradients in INPs. In addition, there is a late INP pulse of Imp. We find that neurons born from old INPs (E-PG and PF-R neurons) have altered morphology following both Imp knock-down and Imp overexpression. We conclude that Imp functions in INPs and newborn neurons to determine proper neuronal morphology and central complex neuropil organization.

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Conflict of interest statement

I am Editor in Chief at Neural Development. A former editor, Julia Kaltschmidt, has agreed to handle our manuscript.

Figures

Fig. 1
Fig. 1
The central complex E-PG and PF-R neurons arise from T2NBs. A The central complex consists of six neuropils: protocerebral bridge (PB, red), fan-shaped body (FB, yellow), ellipsoid body (EB, green), noduli (N, orange), round body (RB, purple), and gall (G, blue). B T2NB neuroblast division pattern. E-PG and PF-R neurons arise from old INPs. C Still frame from Supplemental Video 1. T2NBs identified by pnt-gal4 UAS-GFP and represented as magenta spheres to show position in the 60h ALH central brain. Dorsal view, anterior up. Scale bar 10um. D Maximum intensity projections of confocal imaged PF-R and E-PG neurons in the adult Drosophila brain. Scale bar 20 μm
Fig. 2
Fig. 2
Imp/Syp levels are the same in newborn INPs and T2NBs. A-F T2NBs (cyan circles; Pnt + GFP-) and nINPs (yellow circles, Pnt + GFP-, contacting T2NBs) at 48 h (A), 72 h (B), and 96 h (C). All timepoints have equivalent Imp (A-C) and Syp (D-F) values between T2NBs and nINPs. 12E09 > GFP marks the INP lineage starting at young INP stage. Scale bar 5 μm. G-H Quantification of Imp (G) and Syp (H) fluorescent levels in T2NBs and newborn INPs shows no significant differences at 48 h, 72 h, and 96 h. Each point is a single T2NB or nINP, with all 8 T2NBs included n = 3–5 brains per timepoint. Student t-tests were used to compare T2NBs and nINPs at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. I, K Quantification of average Imp (I) and Syp (J) levels in individual DM1–6 and DL1–2 T2NBs. Note that all T2NBs have a high-low gradient, whereas Syp shows a neuroblast-specific pattern of expression. n = 3–5 brains per timepoint. J T2NBs (cyan circles; Pnt + GFP-) at 48 h and 96 h in DM1 (J) and DM4 (J’). DM1 expresses Syp in a low-to-high expression gradient at 48 h to 96 h. DM4 Syp expression is the opposite high-to-low expression, similar to Imp. Scale bar 5 μm
Fig. 3
Fig. 3
Imp forms a high-low gradient in 48 h INPs. A Quantification of Imp fluorescence in nINPs, yINPs, mINPs, oINPs and nNeurons at 48 h, 72 h, and 96 h. Note that Imp forms a high-low gradient in INPs at 48 h; later timepoints show INP age-specific expression. Each point represents a single INP, n = 3–5 brains per timepoint. ANOVA analysis was used to compare all cell types at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. B-M Confocal images of Imp levels in aging INPs at 48 h (B-E), 72 h (F-I), and 96 h (J-M). See Supplemental Fig. 1 for INP staging criteria. 12E09 > GFP marks the INP lineage beginning at yINPs. Scale bar 5 μm. N Summary
Fig. 4
Fig. 4
Syp forms a high-low gradient in aging INPs. A Quantification of Syp fluorescence in nINPs, yINPs, mINPs, oINPs, and nNeurons at 48 h, 72 h, and 96 h. Syp levels form a high-low gradient in INPs. Each point represents a single INP, n = 3–5 brains per timepoint. ANOVA analysis was used to compare all cell types at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. B-M Confocal images of Syp levels in aging INPs at 48 h (B-E), 72 h (F-I), and 96 h (J-M). See Supplemental Fig. 1 for INP staging criteria. 12E09 > GFP marks the INP lineage beginning at yINPs. Scale bar 5 μm. N Summary
Fig. 5
Fig. 5
16B06-gal4 > ImpRNAi knocks down Imp in oINPs. A 16B06-Gal4 > UAS-GFP UAS-ImpRNAi depletes Imp levels in oINPs, but not T2NBs at 48 h (B), 72 h (C), and 96 h (D). See Supplemental Fig. 1 for INP staging criteria. GFP marks oINPs and nNeurons. Scale bar 5 μm. B-G Confocal images of Imp levels in oINPs (B-D), quantified in E-G. Each point is a single oINPs, n = 3–5 brains per timepoint. H-M Confocal images of Imp levels in nNeurons (E-H), quantified in K-M). Each point is a single nNeuron, n = 3–5 brains per timepoint. Student t-tests were used to compare Imp levels at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Fig. 6
Fig. 6
16B06-Gal4 > ImpOE knocks down Imp in oINPs. A-F 16B06-Gal4 > UAS-GFP UAS-ImpOE depletes Imp levels in oINPs, but not nNeurons at 48 h (A), 72 h (B), and 96 h (C); quantified in D-F. Each point is a single oINPs, n = 3–5 brains per timepoint. G-L 16B06-Gal4 > UAS-GFP UAS-ImpOE increases Imp levels in nNeurons at 48 h (G) and 96 h (I), but not at 72 h (H); quantified in J-L. Each point is a single oINPs, n = 3–5 brains per timepoint. Student t-tests were used to compare Imp levels at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Fig. 7
Fig. 7
ImpRNAi and ImpOE alter E-PG neuropil targeting. A-D Control confocal maximum intensity projection of E-PG neurons (A) and corresponding IMARIS rendering each targeted neuropil (A’-D). Scale bar 20 μm (A-C) or 10 μm (D). E-H ImpRNAi confocal maximum intensity projection of E-PG neurons (E) and corresponding IMARIS rendering each targeted neuropil (E’-H). Scale bar 20 μm (E-G) or 10 μm (H). I-O ImpOE confocal maximum intensity projection of E-PG neurons (I) and corresponding IMARIS rendering each targeted neuropil (I′-O); note that ImpOE results in E-PG neurons generating ectopic projections to the fan-shaped body (FB), noduli (N), and mushroom body (MB). Scale bar 20 μm (J, K, M, N) or 10 μm (L, O). P, Q Quantification of cell numbers (P), and neuropil volume (Q); each point represents an adult Drosophila brain, n = 3–5 brains in control, ImpRNAi, and ImpOE. Student t-tests were used to compare cell numbers to control. ANOVA analysis was used to compare neuropil volumes back to control. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Fig. 8
Fig. 8
ImpRNAi and ImpOE disrupts PF-R neuropil targeting but not cell number. A-D Control confocal maximum intensity projection of PF-R neurons (A) and corresponding IMARIS rendering each targeted neuropil (A’-D). Scale bar 20 μm (A-C) or 10 μm (D). E-H ImpRNAi confocal maximum intensity projection of PF-R neurons (E) and corresponding IMARIS rendering each targeted neuropil (E’-H). Scale bar 20 μm (E-G) or 10 μm (H). I-O ImpOE confocal maximum intensity projection of PF-R neurons (I) and corresponding IMARIS rendering each targeted neuropil (I′-M); note that ImpOE results in PF-R neurons generating ectopic projections to the noduli (N). Scale bar 20 μm (I-K, M) 10 μm (L). N, O Quantification of cell numbers (N), and neuropil volume (O); each point represents an adult Drosophila brain, n = 3–5 brains in control, ImpRNAi, and ImpOE. Student t-tests were used to compare cell numbers to control. ANOVA analysis was used to compare neuropil volumes back to control. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
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