UNC-6/Netrin promotes both adhesion and directed growth within a single axon

Ev L Nichols
Joo Lee
Kang Shen author has email address

Department of Biology, Stanford University, Stanford, United States
Howard Hughes Medical Institute, Stanford University, Stanford, United States
Wu Tsai Neurosciences Institute, Stanford University, Stanford, United States

https://doi.org/10.7554/eLife.100424.2

Open access
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Figures and data

The ventral sublateral nerve cord (vSLNC) is an intermediate target for ventrally growing axons.
A. Diagram of C. elegans illustrating the anteroposterior locations of the neurons which extend ventral axons in the larval stages: AVM, HSN, PDE, PVM, and PQR. B. Confocal time-lapse images of PDE axon guidance. Images were taken every 3 min for 4 hr in the L2 stage. The final image is a confocal still image visualizing the mature PDE morphology in the L4 stage. Blue arrow denotes PDE soma. White text denotes PVD neuron, PDE sheath glia (PDEsh), PDE socket glia (PDEso). Green arrowhead denotes the axonal pause in the sublateral region. Orange arrowhead indicates a dorsal branch, and blue arrowhead denotes a ventral branch. C. Cartoons showing lateral and cross-sectional views of ventral axon navigation in C. elegans. Ventral axons travel along the epidermal edge until they reach the sublateral region. Further ventral growth requires navigation between the epidermis and ventral body wall muscles. D. Electron microscopic image of PDE axon navigation path (blue) in an adult animal. The dashed yellow box denotes the sublateral region where the axonal trajectory changes. This region is enlarged as an inset on the top left. Orange area denotes body wall muscle. Green circle denotes the vSLNC which is ensheathed by the epidermis in late larval stages. E. Confocal images of AVM, HSN, PVM, and PQR axon emergence. The top row visualizes axonal contact with the vSLNC. The bottom row visualizes branching toward the VNC. Dashed green line denotes the vSLNC and the dashed gray line denotes the VNC. White arrow denotes PQR dendrite. F. Confocal images of HSN and PVM axon emergence interacting with the vSLNC. Scale bar is 5 μm in A, E, F and 1 μm in D.

UNC-6 and UNC-40 promote stabilization and directed growth at the sublateral region.
A. Confocal time-lapse images of PDE axons guidance in wild-type, unc-6(ev400), and unc-40(e1430) animals. Images were taken every 3m for 4hr in the L2 stage. Orange arrowhead indicates a dorsal branch, and blue arrowhead denotes a ventral branch. Dashed green line denotes the vSLNC and the dashed gray line denotes the VNC. B. Percentage of axons that stabilize in the sublateral region upon initial contact in wild-type (n=23), unc-6(ev400) (n=18), and unc-40(e1430) (n=21) animals. SE is shown. C. Airyscan images of PDE axon navigation and endogenous UNC-40::GFP growth cone localization in wild-type and unc-6(ev400) animals. Dashed white box denotes growth cone. D. Normalized intensity traces of growth cone UNC-40::GFP during axon navigation normalized to sublateral contact (t=0). Black line denotes wild-type animals (n=8), and red line denotes unc-6(ev400) animals (n=8). SEM is shown. E. Airyscan images of HSN axon emergence and endogenous UNC-40::GFP localization in wild-type and unc-6(ev400) animals. F. Cartoon schematic of polarity measurements and calculation in G. G. UNC-40::GFP polarity in HSN in wild-type (n=20) and unc-6(ev400) (n=21) animals. Positive values denote dorsal polarization, and negative values denote ventral polarization. SEM is shown H. Proportion of branches formed dorsally (orange) and ventrally (cyan) from the sublateral region in wild-type (n=90 branches, 24 animals), unc-6(ev400) (n=114 branches, 25 animals), and unc-40(e1430) (n=114 branches, 28 animals) animals. SE is shown. I. Representative traces of ventral branch extension in wild-type (black), unc-6(ev400) (magenta), and unc-40(e1430) (green) animals. J. Average ventral branch extension speed in wild-type (n=50 branches, 24 animals), unc-6(ev400) (n=43 branches, 25 animals), and unc-40(e1430) (n=32 branches, 28 animals) animals. SEM is shown. K. Percent of axons that stabilize that the VNC within the 4h imaging window in wild-type (n=24), unc-6(ev400) (n=25), and unc-40(e1430) (n=28) animals. SE is shown. Scale bars denote 5 μm in A, C, E. Fisher’s exact test with Bonferonni’s correction is used in B, H, K. Unpaired Student’s t-test is used in G. Ordinary one-way ANOVA with multiple comparisons is used in J. * denotes p<0.05, ** denotes p<0.01, **** denotes p<0.0001. All comparisons to wildtype unless otherwise noted.

SLT-1 directs axon emergence trajectory.
A. Confocal time-lapse images of PDE axons guidance in wild-type, slt-1(eh15), and unc-6(ev400); slt-1(eh15) animals. Images were taken every 3m for 4hr in the L2 stage. Dashed green line denotes the vSLNC and the dashed gray line denotes the VNC. B. Rose plot histogram of PDE axon emergence in slt-1(eh15) (n=24) and unc-6(ev400); slt-1(eh15) (n=26) animals. C. Graph comparing the sine function of axon emergence angle in wildtype (n=22), unc-6(ev400) (n=27), slt-1(eh15) (n=24), and unc-6(ev400); slt-1(eh15) (n=26) animals. 1 indicates 90° dorsal axon emergence and -1 indicates 90° ventral axon emergence. D. Percentage of axons that stabilize in the sublateral region upon initial contact in wild-type (n=23), unc-6(ev400) (n=18), and slt-1(eh15) (n=8), and unc-6(ev400); slt-1(eh15) (n=7) animals. SE is shown. E. Proportion of branches formed dorsally (orange) and ventrally (cyan) from the sublateral region in wild-type (n=90 branches, 24 animals), unc-6(ev400) (n=114 branches, 25 animals), and slt-1(eh15) (n=139 branches, 10 animals), and unc-6(ev400); slt-1(eh15) (n=69 branches, 10 animals) animals. SE is shown. F. Percent of axons that stabilize that the VNC within the 4h imaging window in wild-type (n=24), unc-6(ev400) (n=25), and slt-1(eh15) (n=10), and unc-6(ev400); slt-1(eh15) (n=10) animals. SE is shown. Scale bar denote 5 μm in A. Ordinary one-way ANOVA with multiple comparisons is used in C. Fisher’s exact test with Bonferonni’s correction is used in D-F. Ordinary one-way ANOVA with multiple comparisons is used in C. * denotes p<0.05, *** denotes p<0.001, **** denotes p<0.0001. All comparisons to wildtype unless otherwise noted.

Genetic analysis of PDE axon morphology in adulthood.
“% of axons at VNC” denotes percentage of PDE neurons with axons that extend to the ventral nerve cord (VNC). “% of axons not at VNC” denotes percentage of PDE neurons with axons that do not extend to the ventral nerve cord (VNC). N indicates number of animals scored. P-values generated from Fisher’s exact test.

Genetic analysis of PDE axon morphology in adulthood.
“% of axons at VNC” denotes percentage of PDE neurons with axons that extend to the ventral nerve cord (VNC). “% of axons not at VNC” denotes percentage of PDE neurons with axons that do not extend to the ventral nerve cord (VNC). N indicates number of animals scored. P-values generated from Fisher’s exact test.

Genetic analysis of HSN axon morphology in adulthood.
“% of axons at VNC” denotes percentage of HSN neurons with axons that extend to the ventral nerve cord (VNC). “% of axons not at VNC” denotes percentage of HSN neurons with axons that do not extend to the ventral nerve cord (VNC). N indicates number of animals scored. P-values generated from Fisher’s exact test.

MADD-2 promotes axon stabilization but not growth.
A. Confocal time-lapse images of PDE axons guidance in wild-type and madd-2(ok2226) animals. Images were taken every 3m for 4hr in the L2 stage. Dashed green line denotes the vSLNC and the dashed gray line denotes the VNC. B. Percentage of axons that stabilize in the sublateral region upon initial contact in wild-type (n=23), unc-6(ev400) (n=18), and madd-2(ok2226) (n=11) animals. SE is shown. C. Airyscan images of HSN axon emergence and endogenous UNC-40::GFP localization in wild-type, madd-2(ok2226), madd-2(ok2226); unc-6(ev400), unc-40(4KR); and unc-40(4KR); madd-2(ok2226) animals. D. UNC-40::GFP polarity in HSN in wild-type (n=20), unc-6(ev400) (n=21), madd-2(ok2226) (n=23), and unc-6(ev400); madd-2(ok2226) (n=19) animals. Positive values denote dorsal polarization, and negative values denote ventral polarization. SEM is shown. E. Cartoon schematic of the UNC-40 protein sequence. The dashed red box denotes the intracellular lysine residues that are mutated in unc-40(4KR) animals. F. UNC-40::GFP polarity in HSN in wild-type (n=20), madd-2(ok2226) (n=23), unc-40(4KR) (n=21); unc-40(4KR); madd-2(ok2226) (n=20) animals. Positive values denote dorsal polarization, and negative values denote ventral polarization. SEM is shown. G. Proportion of branches formed dorsally (orange) and ventrally (cyan) from the sublateral region in wild-type (n=90 branches, 24 animals), unc-6(ev400) (n=114 branches, 25 animals), and madd-2(ok2226) (n=52 branches, 12 animals) animals. SE is shown. H. Percent of axons that stabilize that the VNC within the 4h imaging window in wild-type (n=24), unc-6(ev400) (n=25), and madd-2(ok2226) (n=12) animals. SE is shown. Scale bars denote 5 μm in A,C. Fisher’s exact test with Bonferonni’s correction is used in B,G,H. Ordinary one-way ANOVA with multiple comparisons is used in D,F. * denotes p<0.05, *** denotes p<0.001, **** denotes p<0.0001. All comparisons to wildtype unless otherwise noted.

Diffusible UNC-6 is dispensable for axon stabilization.
A. Cartoon schematic of the non-diffusible rescue paradigms at the vSLNC (green, using Pmec-7) and at the VNC (magenta, using Punc-4). B. Confocal time-lapse images of PDE axons guidance in wild-type, unc-6(ev400); Pmec-7::unc-6^TM, unc-6(ev400); Punc-4::unc-6^TM and unc-6(ev400); Pmec-7::unc-6^TM; Punc-4::unc-6^TM animals. Images were taken every 3m for 4hr in the L2 stage. Green lines denote the vSLNC and gray lines denote the VNC. Solid lines denote the location of membrane-tethered UNC-6. C. Percentage of axons that stabilize in the sublateral region upon initial contact in wild-type (n=23), unc-6(ev400) (n=18), unc-6(ev400); Pmec-7::unc-6^TM (n=10), unc-6(ev400); Punc-4::unc-6^TM (n=8) and unc-6(ev400); Pmec-7::unc-6^TM; Punc-4::unc-6^TM (n=7) animals. SE is shown. D. Airyscan images of HSN axon emergence and endogenous UNC-40::GFP localization in wild-type, unc-6(ev400), unc-6(ev400); Pmec-7::unc-6^TM, and unc-6(ev400); Punc-4::unc-6^TM animals. E. UNC-40::GFP polarity in HSN in wild-type (n=20), unc-6(ev400) (n=21), unc-6(ev400); Pmec-7::unc-6^TM (n=25), and unc-6(ev400); Punc-4::unc-6^TM(n=18) animals. Positive values denote dorsal polarization, and negative values denote ventral polarization. SEM is shown. F. Proportion of branches formed dorsally (orange) and ventrally (cyan) from the sublateral region in wild-type (n=90 branches, 24 animals), unc-6(ev400) (n=114 branches, 25 animals), unc-6(ev400); Pmec-7::unc-6^TM (n=105 branches, 14 animals), unc-6(ev400); Punc-4::unc-6^TM (n=83 branches, 10 animals), and unc-6(ev400); Pmec-7::unc-6^TM; Punc-4::unc-6^TM (n=108 branches, 15 animals) animals. SE is shown. G. Percent of axons that stabilize that the VNC within the 4h imaging window in wild-type (n=24), unc-6(ev400) (n=25), unc-6(ev400); Pmec-7::unc-6^TM (n=14), unc-6(ev400); Punc-4::unc-6^TM (n=10), and unc-6(ev400); Pmec-7::unc-6^TM; Punc-4::unc-6^TM (n=15) animals. SE is shown. Scale bars denote 5 μm in B,D. Fisher’s exact test with Bonferonni’s correction is used in C,F, G. Ordinary one-way ANOVA with multiple comparisons is used in E. * denotes p<0.05, ** denotes p<0.01, **** denotes p<0.0001. All comparisons to wildtype unless otherwise noted.

UNC-6 is present in a gradient.
A. Airyscan images of axon emergence in PDE and endogenous UNC-6::mNG distribution in wild-type animals. B. Airyscan images of axon extension in HSN, PDE, and AVM and endogenous UNC-6::mNG distribution in wild-type animals. White arrowheads denote UNC-6 clusters around axonal filopodia. Dashed green line denotes vSLNC and dashed gray line denotes VNC. C. Average intensity of UNC-6::mNG between the VNC (left) and vSLNC (right) in wild-type animals (n=49 animals). C Airyscan images of axon extension in HSN, PDE, and AVM and endogenous UNC-6::mNG distribution in unc-40(e1430) animals. Dashed green line denotes vSLNC and dashed gray line denotes VNC. D. Average intensity of UNC-6::mNG between the VNC (left) and vSLNC (right) in unc-40(e1430) animals (n=37 animals). SEM is shown. Scale bars denote 5 μm in A,C.

UNC-6 clusters are dynamically assembled at the growth cone tip during axon extension.
A. Airyscan time-lapse images of HSN axon extension and endogenous UNC-6::mNG in wild-type and unc-40(e1430) animals. Images were taken every 30s for 30min. White arrowhead denotes the growth cone tip. Dashed white circle denotes UNC-6 clusters on the lateral edges of the growth cone. B. Intensity of UNC-6 foci adjacent to the growth cone membrane in wild-type (n=861 foci, 5 animals) and unc-40(e1430) (n=1306 foci, 5 animals) animals. C. Model of ventral axon migration via alternating haptotaxis and chemotaxis. Scale bar denotes 1 μm in A. Unpaired Student’s t-test is used in B. **** denotes p<0.0001.

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