The conserved RNA-binding protein Imp is required for the specification and function of olfactory navigation circuitry in Drosophila

Abstract

Complex behaviors depend on the precise developmental specification of neuronal circuits, but the relationship between genetic programs for neural development, circuit structure, and behavioral output is often unclear. The central complex (CX) is a conserved sensory-motor integration center in insects, which governs many higher-order behaviors and largely derives from a small number of type II neural stem cells (NSCs). Here, we show that Imp, a conserved IGF-II mRNA-binding protein expressed in type II NSCs, plays a role in specifying essential components of CX olfactory navigation circuitry. We show the following: (1) that multiple components of olfactory navigation circuitry arise from type II NSCs. (2) Manipulating Imp expression in type II NSCs alters the number and morphology of many of these circuit elements, with the most potent effects on neurons targeting the ventral layers of the fan-shaped body (FB). (3) Imp regulates the specification of Tachykinin-expressing ventral FB input neurons. (4) Imp is required in type II NSCs for establishing proper morphology of the CX neuropil structures. (5) Loss of Imp in type II NSCs abolishes upwind orientation to attractive odor while leaving locomotion and odor-evoked regulation of movement intact. Taken together, our findings establish that a temporally expressed gene can regulate the expression of a complex behavior by developmentally regulating the specification of multiple circuit components and provides a first step toward a developmental dissection of the CX and its roles in behavior.

Keywords: RNA-binding proteins; central complex; neural cell fate; neural circuits; neural identity; neural stem cells; neuropeptides; olfactory navigation; temporal patterning; type II lineages.

Figure1.. Overview of central complex structure and development, and olfactory navigation behavioral and circuitry:

A. Drosophila adult brain central complex with labeled neuropils, protocerebral bridge (magenta), fan-shaped body (green), ellipsoid body (orange), and the paired noduli (blue). B. Type II NSCs in the larval brain (blue, 8 per lobe: DM1-6, and DL1-2; central brain-CB, ventral nerve cord-VNC, optic lobe-OL) divide asymmetrically, producing another NSC and an intermediate neural progenitor (INP). The INP divides and gives rise to another INP and a ganglion mother cell (GMC), which eventually forms two neurons and/or glia. Type II NSCs express early and late factors in a time-dependent manner starting from 0h ALH to 120h ALH referred to as early and late factors, respectively. In addition to temporal patterning in NSCs, INPs also express different factors in a birth order-dependent manner along with NSC factors and give rise to a combinatorial code. C. Schematic of the behavioral assay arena; walking flies are observed in a rectangular chamber (14cmX4cm) with constant flow of wind from one direction at 10cm/s. Odor is provided in 10s pulse (1% ACV) centered in a 70s trial. Flies tend to walk upwind in the presence of odor and show search behavior triggered by odor loss. D. CX neurons involved in olfactory navigation investigated in this study. Long-field tangential neurons encode odor, and columnar ventral P-FNs encode wind. Local hΔC/K neurons integrate odor and wind.

Figure 2.. Lineage analysis of olfactory navigation circuit components:

A. Schematic showing 8 Type II NSCs per larval brain lobe (blue) DM1-6 and DL1-2, among other NSCs (Type 0 and Type I) in red. Central brain-CB, ventral nerve cord-VNC, optic lobe-OL. B. Genetic scheme for Type II lineage analysis using Type II specific Flip-out. Ase-GAL80 active in Type I NSCs leaves Wor-GAL4 active only in Type II NSCs. Flippase downstream of a UAS sequence is activated in a Type II-specific manner and removes the STOP sequence between two FRT sites. This causes expression of reporter GFP in class-specific manner when crossed to a class-specific LexA driver line. C-F. Confocal images showing neuron types expressing reporter GFP in a Type II dependent manner. G. Genetic scheme for Type II lineage filtering using the CLIn technique. A Type II specific promoter fragment (stg) combined with KD recombinase removes a STOP sequence by a flip event and leaves Cre recombinase active in Type II NSCs. Cre generates Type II NSC clones positive for LexA::p65. GAL80 is active in the rest of the cells, and GAL4 expresses GFP as a reporter in class specific manner. We did not stain for mCherry here. H-I. Confocal images showing neuron types expressing reporter GFP in a Type II dependent manner. nc82 stain in magenta counterstains the brain, a stack of 2 slices was taken for visualizing the neuropil with nc82. GFP reporter is shown in green. Cell bodies are indicated by yellow arrows. The genotypes are shown at the top, and the abbreviated genotypes for neuron class are shown at the left. Scale bars correspond to 30μm. See also Figure S1, Table 1 and Video S1

Figure 3.. Birth-dating vFB long field tangential input neurons using cell class lineage intersection system:

A. Schematics describing the genetics of the CLIn system combined with a heat shock promoter for genetic birth-dating: Type II specific stg promoter fragment combined with KD recombinase removes a STOP sequence by a flip event, and an intact Flippase is reconstituted. This combined with heat-sensitive promoter removes STOP sequence and leaves Cre-recombinase active in Type II NSCs in a heat shock dependent manner. Cre in turn generates Type II NSC clones positive for LexA::p65. GAL80 is active in the rest of the cells, and GAL4 expresses GFP as a reporter in class-specific manner, while mCherry downstream of LexAop sequence is expressed as a reporter in a lineage-specific manner. B. Schematics representing heat shock given at different time points (ALH-after larval hatching), yellow arrow indicates heat shock, and GFP expression window is shown in green. C. Schematics showing the map of the progeny of the DL1 clone populating dorsal and ventral layers of the FB in the adult brain. D. Adult brain sample of 0h ALH DL1 clone of CLIn crossed to VT029515 GAL4 labeling vFB neurons, nc82 counterstains the brain, a stack of 2 slices was taken for visualizing the neuropil with nc82. DsRed in cyan labels the DL1 progeny, GFP in green labels vFB neurons. E-H. Adult brain samples of 0h, 24h, 48h, and 72h ALH clones, GFP in green labels vFB neurons. I. Quantification of number of cell bodies counted per hemibrain at indicated time points (n=data points indicated on the graph). J.17A12-GAL4 flipped vFB neurons, nc82 counterstains the brain, a stack of 2 slices was taken for visualizing the neuropil with nc82. GFP in green labels vFB neurons. K. Quantification of number cell bodies per hemibrain flipped by 17A12-GAL4.The genotypes are shown at the top, and the abbreviated genotypes for neuron class are shown at the left. Scale bars correspond to 30μm. See also Figure S1, Video S1 and S3

Figure 4.. Imp is required for specifying and maintaining the identity of odor encoding vFB long field input neurons:

A-C’. Effects of Type II-specific knockdown (Pointed-GAL4>ImpRNAi; ImpRNAi) or overexpression (Pointed-GAL4>UAS-Imp) of Imp on vFB neurons. vFB neurons labeled by reporter GFP in control (A), Imp knockdown (B), and Imp overexpression (C), nc82 counterstains the brain, and GFP is shown in green. A’’-C’’. vFB neuron arbors shown by GFP expression in green, neuropeptide TK staining shown in cyan colocalizing with GFP for control (A’’), Imp knockdown, expression observed in dorsal layers of FB (B’’) and Imp overexpression, TK expressed in thick bundle colocalizing with GFP expression (C’’). D. vFB neurons labeled with GFP via conditional expression of GFP using TK-GAL4, nc82 counterstains the brain. Zoomed in view of vFB cell bodies, GFP in green (D’), and TK expression in magenta (D’’), (n=6). E-E’. Effects of DL1/2 specific knockdown of Imp on vFB neurons. vFB neurons labeled by reporter GFP are shown in green in control (E) and Imp knockdown (17A12-GAL4>ImpRNAi; ImpRNAi) (E’), nc82 counterstains the brain. F-G’’. Effects of post-mitotic knockdown of Imp on vFB neurons. vFB neurons labeled by reporter GFP are shown in green, Imp expression is shown in magenta in control (F-F’’) and Imp knockdown (vT029515-GAL4>ImpRNAi; ImpRNAi) (G-G’’). For F’-F’’ and G’-G’’, the Scale bar corresponds to 15μm. Quantification of the number of cell bodies per hemibrain for Type II specific knockdown (H), DL1 specific knockdown (I), and post-mitotic knockdown (J). n=data points indicated on the graphs (Students t-test). The abbreviated genotypes are shown at the left and top, respectively. Scale bars correspond to 30μm (unless stated otherwise). The dashed outline shows FB in A-C’’ and cell bodies in D’-D’’ and F’’ and G’’. See also Figures S1, S2, S3, S4 and Table 1

Figure 5.. Imp regulates specification and morphology of other neural types in the olfactory navigation circuit:

A-C. Ventral P-FN neurons labeled by reporter GFP shown in control (A), Imp knockdown (Pointed-GAL4>ImpRNAi; ImpRNAi) (B), and Imp overexpression (Pointed-GAL4>UAS-Imp) (C). D-F. dFB (65C03) neurons labeled by reporter GFP shown in control (D), Imp knockdown (Pointed-GAL4>ImpRNAi; ImpRNAi) with defective morphology of the neurites in FB (E), and Imp overexpression (Pointed-GAL4>UAS-Imp) (F). G-I. FB5AB input neurons labeled by reporter GFP in control (G), Imp knockdown (Pointed-GAL4>ImpRNAi; ImpRNAi) with defective morphology of the neurites in FB (H), and Imp overexpression (Pointed-GAL4>UAS-Imp) (I). J-L. hΔC/K neurons labeled by reporter GFP in control (J), neurites arborize multiple layers of FB with no clear demarcation in Imp knockdown (Pointed-GAL4>ImpRNAi; ImpRNAi) (K) and Imp overexpression (Pointed-GAL4>UAS-Imp) (L). GFP is shown in green, the abbreviated genotypes are shown at left and top. Scale bars correspond to 30μm. M-P. Quantification of number of cell bodies. Abbreviated neuron names are indicated at the top. n=data points indicated on the graphs. (Students t-test). Q-U’. The four neuropils of the CX: PB, NO, FB, and EB in control flies (Q-T), and Imp knockdown resulting in defective morphology of the CX neuropils (Q’-T’). nc82 stains the neuropils of the brain, all outlined using dashed lines, (n=22). U-U’. sNPF stains shown in cyan in control distributed in dorsal and ventral layers of FB distinctly (U), and Imp knockdown shows impaired distribution (U’). Scale bars correspond to 30μm. See also Figures S2, S4 and Videos S2 and S3

Figure 6.. Imp regulates the upwind orientation during olfactory navigation:

A. Representative walking trajectories of three different control flies (Pointed-GAL4>UASmCherryRNAi) presented with a 10s odor pulse (1% ACV, magenta) centered in a 70s trial with 10cm/s wind. Flies demonstrate upwind movement during odor and local search after odor offset. Wind direction indicated above. B. Representative walking trajectories of three different Imp knockdown flies (Pointed-GAL4>UAS-ImpRNAi;UAS-ImpRNAi) in the same wind and odor conditions as Figure A. Trajectories reveal lack of upwind movement during odor and increased turning behavior throughout the entirety of trial. C. Comparison of movement parameters between control (black, n = 86 flies) and Imp knockdown flies (red, n = 61 flies) calculated as an average across flies (mean±SEM; see Methods). Pink-shaded area: 10-second odor stimulation period (1% ACV). In the presence of odor, Imp knockdown flies show decreased upwind velocity comparable to control flies. Increased turning behavior noted in Figure B can be observed quantitatively through the increased angular velocity throughout the duration of trials. D. Heading distribution during odor for both control (black) and Imp knockdown (red) flies (wind direction indicated above). Imp knockdown flies exhibit decreased upwind orientation. E. Histogram showing ground speed distributions during odor for both control (black) and Imp knockdown (red). Distributions appear similar, suggesting that general movement is not impaired by Imp knockdown. F. Histogram showing angular velocity distributions for both control (black) and Imp knockdown flies(red) during odor. Imp knockdown flies favor larger angular velocities, even in the presence of an attractive stimulus. G. Heading distribution during odor for both control (black) and knockdown of Imp (red) in DL1/2 Type II NSCs (wind direction indicated by above arrow). H. Histogram showing ground speed distribution during odor for both control (black) and Imp knockdown (red) flies. Imp knockdown flies tend towards lower ground speeds than control counterparts. I. Histogram showing angular velocity distribution during odor for both control (black) and Imp knockdown (red) flies. Widening of distribution indicates that Imp knockdown flies favor larger angular velocities. J. Change in upwind velocity and ground speed, calculated as the difference in the mean value during the first 5 seconds of odor and the 5 seconds preceding odor, across various control and experimental genotypes. Knockdown of Imp results in significant decreases in upwind velocity change when compared to respective controls, across multiple odor strengths and driver lines (Pointed-GAL4>mCherryRNAi compared to pointed>double ImpRNAi p = 5.906x10^-11, Pointed-GAL4>mCherryRNAi compared to Pointed-GAL4>ImpRNAi(III) p=1.14768x10^-9, Pointed-GAL4>mCherryRNAi (1:10 AVC) compared to Pointed-GAL4>double ImpRNAi (10:1) p=1.2293x10^-4, DL1>mCherry compared to DL1>double ImpRNAi p=9.922x10^-6). Knockdown of late transcription factor Syncrip (Syp) results in a significant increase in upwind velocity when using a higher odor concentration (1:10 ACV, Pointed-GAL4>mCherryRNAi compared to Pointed-GAL4>SypRNAi (10:1) p=0.0042). Knockdown of Imp using a single copy of the RNAi results in a significant decrease in ground speed (Pointed-GAL4>mCherryRNAi compared to Pointed-GAL4>ImpRNAi (III) p=0.0015), as does knockdown in DL1/DL2 Type II NSCs (17A12-GAL4> ImpRNAi;ImpRNAi ,two-sample t-test; p =2.2281x10^-7). Upregulation of Imp results in increased ground speed but no change to upwind velocity (Pointed-GAL4>mCherryRNAi compared to Pointed-GAL4>UAS-Imp p=7.7113x10^-4). All comparisons were completed using an unpaired t-test with Bonferroni correction for multiple comparisons. See also Figures 4, S1 and S2