Spatiotemporal Changes in Netrin/Dscam1 Signaling Dictate Axonal Projection Direction in Drosophila Small Ventral Lateral Clock Neurons

Jingjing Liu
Yuedong Wang
Xian Liu
Junhai Han author has email address
Yao Tian author has email address

School of Life Science and Technology, the Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096, China
Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China

https://doi.org/10.7554/eLife.96041.2

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Figures and data

s-LNvs axon projection dynamics during the larval stage.
(A) Images showing the growth process of s-LNvs during larval development. Larval brains were stained with anti-PDF (white) and HRP (magenta) antibodies. Different time points indicate the hours after larval hatching (ALH).
(B) Left panel: schematic diagram illustrating the method used to measure the degree of vertical projection. One hemisphere of the larvae brain is depicted. larval neuropil (gray), s-LNvs (green), optic lobe (blue). Right panel: graphs showing the average vertical projection length at different developmental stages, presented as mean ± SD.
(C) Left panel: schematic diagram illustrating the method used to measure the degree of horizontal projection. Right panel: graphs showing the average horizontal projection (A.U.: arbitrary unit) at different developmental stages, presented as mean ± SD. The red line segment indicates the stage at which the axonal projection undergoes a directional transition.
(D) Schematic representation of s-LNvs vertical to horizontal projection directional shift during 72-96 hours ALH. One hemisphere of the larvae brain (gray), larval neuropil (white), s-LNvs (green).
For (B), (C), 8 h (n = 7), 24 h (n = 10), 48 h (n = 14), 72 h (n = 16), 96 h (n = 13), 120 h (n = 16).

Development of vertical projection process alongside Mushroom Body growth.
(A) Spatial position relationship between calyx and s-LNvs. Top: OK107-GAL4 labels the calyx of MB γ and α’β’ neurons, while s-LNvs are labeled by the PDF antibody. Bottom: Tab2-201Y-GAL4 labels calyx of MB γ neurons, with s-LNvs labeled by the PDF antibody. Larvae brains were stained with anti-PDF (green) and GFP (magenta) antibodies. Different time indicated hours ALH. The white dotted line indicates the calyx region.
(B) Graphs showing the mushroom body calyx’s area and vertical projection length averaged over different development stage are presented as mean ± SD. OK107: 24 h (n = 10), 48 h (n = 8), 72 h (n = 12), 96 h (n = 12), 120 h (n = 9). Pearson’s r = 0.9987, P < 0.0001. Tab2-201Y: 24 h (n = 6), 48 h (n = 12), 72 h (n = 13), 96 h (n = 13), 120 h (n = 11). Pearson’s r = 0.9963, P = 0.0003.
(C) Schematic representation of complementary development of s-LNvs vertical projection and mushroom body calyx. Mushroom body (purple), s-LNvs (Green).
(D) Images of mushroom body ablation in the developing larval stages. Larvae brains were stained with anti-PDF (white) and HRP (blue) antibodies. Different time indicated hours ALH.
(E) Quantification of vertical projection length in Control (Tab2-201Y >GFP) and Ablation (Tab2-201Y >GFP,rpr,hid) flies. Data are presented as mean ± SD. Control: 24 h (n = 6), 48 h (n = 14), 72 h (n = 13), Ablation: 24 h (n = 6), 48 h (n = 12), 72 h (n = 12). Two-tailed Student’s t tests were used. ns, p > 0.05; ****p < 0.0001.

Critical role of Dscam1 in s-LNv axonal horizontal projection.
(A) Images of s-LNvs in Pdf >GFP and Pdf >GFP, Dscam1^RNAi fly. White line segment represents the horizontal projection distance of s-LNvs. Larvae brains were stained with anti-PDF (white) and HRP (blue) antibodies at 96 hours ALH. Pdf >GFP: (n = 13), Pdf >GFP, Dscam1^RNAi: (n = 15).
(B) Quantification of vertical projection length in Pdf >GFP and Pdf >GFP, Dscam1^RNAi flies. Data are presented as mean ± SD. Two-tailed Student’s t tests, ns, p > 0.05.
(C) Quantification of horizontal A.U. in Pdf >GFP and Pdf >GFP, Dscam1^RNAi flies. Data are presented as mean ± SD. Two-tailed Student’s t tests, ****p < 0.0001.
(D) Images of Dscam1 mutant s-LNvs projection phenotype. Larvae brains were collected at late 3rd larvae. Heads were stained with anti-PDF (white) and HRP (blue) antibodies.
(E) Quantification of horizontal A.U. in Dscam1 mutant flies. Data are presented as mean ± SD. w¹¹¹⁸ (n = 22) Dscam1²¹/+ (n = 5), Dscam1¹/+ (n = 8), Dscam1⁰⁵⁵¹⁸/+ (n = 10), Dscam1²¹/ Dscam1⁰⁵⁵¹⁸(n = 6), Dscam1²¹/Dscam1¹ (n = 10), Dscam1⁰⁵⁵¹⁸(n = 14). One-way ANOVA with Tukey’s post hoc, ns, p > 0.05, ****p < 0.0001.
(F) Endogenous Dscam1 co-localizes with the s-LNvs axon terminal. Pdf >GFP fly heads were collected at 120 h ALH and stained with anti-Dscam1 (red). The white dotted line indicates the s-LNvs axon.

Neuron-derived Netrin guides s-LNvs horizontal projection.
(A) Images of immunostained in w¹¹¹⁸, NetA^Δ, NetB^Δ and NetAB^Δ fly. Larvae brains were collected at late 3rd larvae. Heads were stained with anti-PDF (white) and HRP (blue) antibodies.
(B) Quantification of horizontal A.U. w¹¹¹⁸, NetA^Δ, NetB^Δ and NetAB^Δ fly. Data are presented as mean ± SD, w¹¹¹⁸ (n = 17), NetA^Δ (n = 15), NetB^Δ (n = 24), NetAB^Δ (n = 23). One-way ANOVA with Dunnett’s post hoc, ns, p > 0.05, ***p < 0.001.
(C) Images of immunostained in nSyb-GAL4 and repo-GAL4 knockdown Netrins. Larvae brains were collected at late 3rd larvae heads were stained with anti-PDF (white) and HRP (blue) antibodies.
(D) Quantification of horizontal A.U. in nSyb-GAL4 and repo-GAL4 knockdown Netrins fly. Data are presented as mean ± SD. nSyb-GAL4/+ (n = 13), nSyb-GAL4 >Netrins^RNAi (n = 16), repo-GAL4/+ (n = 34), repo-GAL4 >Netrins^RNAi (n = 20). Two-tailed Student’s t tests, ns, p > 0.05, ***p < 0.001.

Netrin secreted by DN neurons guides time-specific horizontal projection of s-LNvs.
(A) Images of immunostained in OK107-GAL4 and per-GAL4, Pdf-GAL80 knockdown Netrins. Larvae brains were collected at late 3rd larvae. Heads were stained with anti-PDF (white) and HRP (blue) antibodies.
(B) Quantification of horizontal A.U. in OK107-GAL4 and per-GAL4, Pdf-GAL80 knockdown Netrins fly. Data are presented as mean ± SD. OK107-GAL4/+ (n = 20), OK107-GAL4 >Netrins^RNAi (n = 13). per-GAL4, Pdf-GAL80/+ (n = 18), per-GAL4, Pdf-GAL80 >Netrins^RNAi (n = 16), Two-tailed Student’s t tests for OK107-GAL4 knockdown Netrins, ns, p > 0.05. Mann-Whitney test for per-GAL4, Pdf-GAL80 knockdown Netrins, ****p < 0.0001.
(C) Images of DN ablation in the developing larval stages. Larvae brains were stained with anti-PDF (white) and HRP (blue) antibodies. Different time indicated hours ALH.
(D) Quantification of horizontal A.U. in Control (per-GAL4, Pdf-GAL80 >GFP) and Ablation (per-GAL4, Pdf-GAL80 >GFP,rpr,hid) flies. Data are presented as mean ± SD. Control: 72 h (n = 5), 96 h (n = 12), Ablation: 72 h (n = 5), 96 h (n = 10). Two-tailed Student’s t tests were used to compare conditions. ns, p > 0.05, ****p < 0.0001.
(E) Images of immunostained in per-GAL4, Pdf-GAL80 overexpress NetB in NetAB^Δ. Larvae brains were collected at late 3rd larvae. Heads were stained with anti-PDF (white) and HRP (blue) antibodies.
(F) Quantification of horizontal A.U. in per-GAL4, Pdf-GAL80 overexpress NetB in NetAB^Δ. Data are presented as mean ± SD. NetAB^Δ, UAS-NetB (n = 12), NetAB^Δ, per-GAL4,Pdf-GAL80 /+ (n = 5), NetAB^Δ,per-GAL4,Pdf-GAL80 >UAS-NetB (n = 9). One-way ANOVA with Bonferroni post hoc, ns, p > 0.05, ***p < 0.001.

Dynamic changes in DN neurons coordinated with s-LNvs axonal targeting.
(A) Top: Images of s-LNvs axon and DNs growth process in the developing larval stages. Larvae brains were stained with anti-PDF (white) and GFP (green) antibodies. Different time indicated hours ALH are shown. Bottom: schematic diagram of s-LNvs horizontal projection and the corresponding increase in the number of DN neurons. s-LNvs axon (gray), DN neurons (green).
(B) Quantification of the number of DN neurons labeled by per-GAL4, Pdf-GAL80 at different developmental stages. Data are presented as mean ± SD.
(C) Images of newborn DN neurons were colocalized with Netrin-B at different developmental times. Larvae brains were stained with anti-GFP (green), mcherry (red) and PDF (white) antibodies. Different time indicated hours ALH. The white arrow demarcates the co-localization of red and green signals.
(D) Schematic representation s-LNvs projection directional transition and the corresponding increase in the number of DN neurons. s-LNvs axon (gray), DN neurons (green), newborn DN neurons (light green at 72 h, orange at 96 h).

Dynamic cellular molecular environment during the s-LNv projection directional shift.
Cartoon depicting the dynamic cellular molecular microenvironment during the axonal projection directional shift from vertical to horizontal projection in s-LNvs. 72 h and 96 h represent 72 h and 96 h after larval hatching, respectively. s-LNvs (blue), neuropile (pink), brain lobe (gray circule), DNs (orange and light orange circles), optic lobe (gray dotted line), Netrin (Orange combination molding), Dscam1 (pruple).

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