Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila melanogaster CNS

Abstract

Neurotrophin receptors corresponding to vertebrate Trk, p75(NTR) or Sortilin have not been identified in Drosophila, thus it is unknown how neurotrophism may be implemented in insects. Two Drosophila neurotrophins, DNT1 and DNT2, have nervous system functions, but their receptors are unknown. The Toll receptor superfamily has ancient evolutionary origins and a universal function in innate immunity. Here we show that Toll paralogs unrelated to the mammalian neurotrophin receptors function as neurotrophin receptors in fruit flies. Toll-6 and Toll-7 are expressed in the CNS throughout development and regulate locomotion, motor axon targeting and neuronal survival. DNT1 (also known as NT1 and spz2) and DNT2 (also known as NT2 and spz5) interact genetically with Toll-6 and Toll-7, and DNT1 and DNT2 bind to Toll-6 and Toll-7 promiscuously and are distributed in vivo in domains complementary to or overlapping with those of Toll-6 and Toll-7. We conclude that in fruit flies, Tolls are not only involved in development and immunity but also in neurotrophism, revealing an unforeseen relationship between the neurotrophin and Toll protein families.

Fig.1. Toll-6 and Toll-7 are expressed in the CNS through all stages

In situ hybridisations showing transcripts for Toll-6 and Toll-7 in: (a,b) stage 13, 15 and 17 embryos; (e,g) larval optic lobes, central brain and ventral nerve cords; (i,k) adult brain in central complex (arrows). (c,d,f,h,j,l) Distribution of GFP in Toll6^MIMICGFP and anti-Toll7 matches that of the transcripts. (c) Note GFP signal in distinct neuronal types (arrows); (d) Note motoraxons exiting the CNS (first and second image, white arrows), motorneuron cell bodies (yellow arrowheads), axons crossing the midline (third image, arrows), and along three interneuron fascicles (fifth image, arrows), and in thickenings that might correspond to dendrites or glia (yellow arrowhead). (i,k) Note signal in and around fan shaped body. EL, U/CQs: Eve neurons. Anterior is up. Scale bar in: (a,c) 10 μm; (f,h,j,l) 50 μm.

Fig. 2. Identification of Toll-6 and Toll-7 cells in the locomotor circuit

(a,c,e) Anti-GFP in Toll6^MIMICGFP embryos is distributed in ventral lateral nerve cord HB9+ neurons (a), Eve+ EL interneurons and all Eve+ motorneurons except RP2 (c,e, arrows, for RP2s see Supplementary Fig.2c). IN=interneurons, arrowheads pointing at longitudinal connectives. (b,d,f) Toll-7 protein is localised to ventral lateral and medial HB9+ neurons (b, arrows), Lim3GAL4>myrRFP+ RP motorneurons (d, arrows) and possibly dendrites (pink arrow) and FasII+ interneuron fascicles (f, arrows). (g) Toll-6GAL4 (D42)>10xmyr-tdTomato and Toll-7GAL4>GAPGFP reveal ISNd/b terminals (compare to Lim3GAL4>myrRFP). (h) Toll6^MIMICGFP and Toll-7 are present in the larval VNC neuropile (arrows pint at axons) and at least Toll6^MIMICGFP in motorneurons. (i) Toll6^MIMICGFP colocalises with the dopamine precursor Tyrosine Hydroxylase (TH) in most dopaminergic neurons. (j) Toll6^MIMICGFP and Toll-7 are distributed in distinct layers of the fan-shaped body (fsb, arrows) and rings of the ellipsoid body (eb, arrows). Scale bars: (a-g) 10μm, (h-j) 50μm.

Fig. 3. Toll-6 and Toll-7 are required for larval locomotion and motor-axon targeting

(a) Trajectories of larvae crawling for 400 frames per larva, n=50 larvae per genotype . (b) Toll-7 Toll-6 double mutant larvae crawl more slowly. Kruskal-Wallis 814: p<0.0001, and Dunn’s test for pair-wise comparisons, asterisks refer to double mutants vs. yw controls (Dunn=4474), n=50 larvae and 19950 frames per genotype. (c-e) The incidence of FasII+ motoraxon misrouting in 1 or more projections, and loss of two or more projections, per hemisegment increase in (c,e) stage 17 mutant embryos and (d,e) embryos over-expressing activated forms of Toll-6 or Toll-7 in all neurons (elavGAL4>Toll-6^CY;Toll-6^CY and Toll-7GAL4;elavGAL4>Toll-7^CY). Scale bar: 10 μm. (e) Chi-Square χ² (7)=136.247 p<0.001, pair-wise comparisons to yw Chi-square with Bonferroni correction, n=169-465 hemisegments per genotype. *p<0.05; ** p<0.01; ***p<0.001. For further details see Supplementary Table 3.

Fig. 4. Toll-6 and Toll-7 maintain neuronal survival

(a) Embryonic VNCs labelled with anti-cleaved-Caspase-3. (b,c) Apoptosis increases in Toll-7 and Toll-6 mutant embryos, as quantified with DeadEasy software: (b) One Way ANOVA F(2,70)=5.782: p=0.005, post-hoc Dunnett p=0.006, p=0.015, respectively, n=19-28 embryos per genotype; (c) One Way ANOVA F(2,71)=7.010 p=0.002, post-hoc Dunnett p=0.001, p=0.032, respectively, n=21-31 embryos. (d) Pan-neuronal over-expression of activated Toll-6 and Toll-7 rescues naturally occurring cell death in the CNS, One Way ANOVA F(2,68)=4.811 p=0.011, post-hoc Dunnett p=0.021, p=0.012, respectively, n=22-27 embryos. (b-d) Asterisks refer to pair-wise comparisons to yw, post-hoc Dunnett tests. (e-h) Apoptotic Caspase+HB9+ cells in Toll7^P8/Toll7^P114; Toll6²⁶/Toll6³¹ double mutant embryos in locations corresponding to neurons that normally express (e) Toll-6 or (f) Toll-7, (g) high magnification view and (h) quantification, unpaired Student t-test (1)=−2.230, p=0.035, n=9-19 embryos. (i,j) In Toll7^P8/Toll7^P114; Toll6²⁶/Toll6³¹ double mutant embryos, more EL clusters have Eve+ Caspase+ apoptotic interneurons (j, albeit not significant χ²(1)=1.992 p=0.158, n=109-138 EL clusters). (k,l) Apoptosis leads to loss of Eve+ EL interneurons in the double mutants, as more EL clusters have fewer neurons than the normal 8-10 per cluster (arrows in k), Chi-square χ²(1)=9.645 p=0.002, n=22-260 EL clusters. *p<0.05; **p<0.01;***p<0.001. All stage 17 embryos. Scale bars: (a) 20 μm; (e, f, k) 10 μm; (g, i) 5 μm. For further details see Supplementary Table 3.

Fig. 5. Toll-6 and Toll-7 interact genetically with DNT2 and DNT1

Survival index for homozygous yw;;+/+ controls bred from an outcross to TM6B at 18°C is 1. (a) Single homozygous mutants lacking one DNT or Toll-6 or Toll-7 are viable, whereas homozygous double mutants lacking DNT1 and DNT2 or Toll-6 and Toll-7 are semi-lethal if bred at 18°C as progeny of a stock maintained over a TM6B or SM6aTM6B balancer. Chi-square χ²(11)=360.277 p<0.001, n=126-872 pupae per genotype. (b) The semi-lethality of Toll-7^P8;Toll-6²⁶ double mutants can be rescued by over-expressing the activated receptors in cholinergic neurons. χ²(6)=85.028 p<0.001, n=102-467 pupae. (c) Homozygous double mutants lacking one DNT and one Toll recapitulate the semi-lethality of DNT1⁴¹DNT2^e03444 and Toll-7^P8;Toll-6²⁶ double mutants, and the lethality DNT1 Toll-6 double mutants can be rescued by expressing the activated receptors with Toll-7GAL4. χ²(5)=653.525 p<0.001, n=72-991. (d) The semi-lethality of DNT1⁴¹DNT2^e03444 double mutants can be rescued by expressing the activated Toll-6, Toll-7 or Toll receptors in neurons. χ²(10)=401.419 p<0.001, n=83-1461 pupae. (e,f) Quantification of anti-cleaved-Caspase-3 labelling in embryonic VNCs: apoptosis increase of DNT⁵⁵ and DNT2^e03444/Df6092 mutant embryos (e, One Way ANOVA F(2,69)=10.479 p<0.001, post-hoc Dunnett p<0.01, p=0.051) is rescued with the over-expression of activated Toll-7^CYand Toll-6^CY in all neurons (f, Welch ANOVA F(2,63)=5.143 p=0.009, post-hoc Dunnett p=0.011, p=0.017). (e,f) Asterisks refer to pairwise comparisons to yw, post-hoc Dunnett tests. (g) Pan-neuronal over-expression of activated Toll^10b, Toll-6^CY and Toll-7^CY rescues the semi-lethality of spz² mutants, χ²(7)=99.272 p<0.001. ***p<0.001; **p<0.01; *p<0.05. (a-d,g) Asterisks refer to Chi square comparisons to fixed controls with Bonferroni corrections. For detailed genotypes and further statistics details, see Supplementary Tables 1 and 3.

Fig. 6. In vitro, cell culture and in vivo evidence that Toll-7 and Toll-6 bind DNT1 and DNT2

(a) Diagrams illustrating the constructs encoding tagged proteins. (b) Coomassie stainings showing: Left: secreted Toll-6/7ECD purified from S2 cell conditioned medium, for mass spectrometric sequence evidence see Supplementary Fig.5e. Centre: DNT2 is purified from Baculovirus as a secreted cleaved cystine-knot (CK) dimer. Right: DNT1 is purified from S2 conditioned medium as cleaved 45kDa cystine-knot plus terminal extension (CK+CTD, lower arrow), also in full-length (FL) form and cleavage products (upper arrow), and DNT2 is purified from S2 conditioned medium only as cleaved cystine-knot. (c) The mass of DNT2 purified by reverse phase chromatography determined by MALDI TOF mass spectrometry demonstrates that DNT2 is secreted as a cleaved cystine-knot. The observed mass of the DNT2 dimer was 26225.82±0.62 Da, matching exactly the expected mass. DNT2 is cleaved at a trypsin-like cleavage site. In contrast to Spz, but like vertebrate NGF, DNT2 is processed during biosynthesis. (d) Native gel showing complexes of purified DNT2CK with purified Toll-6ECD, Toll-7ECD or both Toll-6ECD+Toll-7ECD: the shift in the Toll-6/7 band when mixed with DNT2 indicates that these proteins interact (western blot, anti-His). (e) Predicted mobility of native Toll-6ECDHisFLAG, Toll-7ECDHisFLAG and DNT2CKHis at pH=8.8. At this pH the charge of DNT2 is very close to 0, whereas Toll-6 and Toll-7 are negatively charged. (f) Co-transfection S2 cell lysate controls for ELISA and co-IP experiments showing proteins expressed in each experiment in (g,h,i). (g) ELISA assays using co-transfected S2 cells revealed a significant difference in absorbance comparing single and co-transfected S2 cell lysates. Unpaired t-tests: top 6 vs 2: t(4)=−10.485 p<0.001; 7 vs 3: t(4)=−7.619 p=0.002; bottom 7 vs 4: t(4)=−5.574 p=0.005, 6 vs 5: t(4)=−13.504 p<0.001, n=3 repeats. (h,i) Co-immunoprecipitation of full-length Toll-7HA and DNT1V5, and full-length Toll-6HA and DNT2V5, from co-transfected S2 cells. (h) Precipitation of receptors with anti-HA brings down bound ligands detected with anti-V5; (i) precipitation of ligands with anti-V5 brings down bound receptors detected with anti-HA. (j) In vivo co-immunoprecitation from transgenic flies over-expressing full-length Toll-7HA and DNT1-FLAG in the retina with GMRGAL4. Two examples are shown, using rabbit (left) or mouse (right) anti-FLAG antibodies to precipitate DNT1, bringing down bound receptor detected with anti-HA. ***p<0.001, **p<0.01, *p<0.05. (h, i, j) Co-IP blots have been cropped for clarity; full-length blots are shown in Supplementary Figs.6-9.

Fig. 7. The relative distributions of DNT1, 2 and Toll-7, 6, respectively, in vivo are consistent with their functions are ligand-receptor pairs

(a) Anti-DNT1 reveals DNT1 protein distributed in the embryonic CNS midline (stage 15) and (b) at high levels in muscle 13,12, in lower levels in muscles 6,7 and possibly others too (stage 17). (c,d) Anti-DNT2 reveals punctate signal along larval CNS axons revealed with (c) FasII+, (d) DsRed+ in Toll-6GAL4(D42)>myrRFP and (e) Toll-6^MIMICGFP. (f) Anti-DNT1 and anti-Toll-7 co-localise in fan-shaped body layers. Anti-DNT2 and anti-GFP in Toll-6^MIMICGFP are distributed in complementary fan-shaped body layers. Anterior is up, scale bar: (a,d,e) 10μm, (c,f) 50μm.