Segment-specific axon guidance by Wnt/Fz signaling diversifies motor commands in Drosophila larvae
- Department of Complexity Science and Engineering, Graduate School of Frontier Sciences, The University of Tokyo, Japan
- Department of Physics, Graduate School of Science, The University of Tokyo, Japan
Figures
Figure 1 with 3 supplements
DFz2 inhibits Wave axon extension toward the posterior end.
(A, A’) Schematic diagram of Wave neuron morphology and function. Wave neurons are segmentally repeated in the ventral nerve cord (VNC), receive inputs from tactile sensory neurons, and drive distinct behaviors depending on the segment. The anterior Wave (a-Wave) comprises anteriorly polarized axon/dendrite and drives backward locomotion, whereas posterior Wave (p-Wave) has axon/dendrite extension toward the posterior end and drives forward locomotion. (B–E) Lateral view of single-cell images of Wave neurons revealed by mosaic analyses in 3rd instar larvae. The green channel shows Wave neurons, and the blue channel shows anti-HRP staining that visualizes the neuropil. SEZ: subesophageal zone, T1-3: thoracic VNC, A1-8/9: abdominal VNC. (B, C) Control A2 (B) and A6 (C) Wave neuron. Scale bar = 50 µm. White arrows indicate axon processes. (D, E) A2 (D) and A6 (E) Wave neuron in which DFz2 is knocked down using the TRiP RNAi (#67863) line. Note the elongation of the axons toward the posterior end (dotted boxes). Orange arrows indicate the putative presynaptic varicosities in the ectopic axons. (F–J) Tiling profile of the axons of A2 to A6 Wave neurons in control and DFz2 knocked-down animals. n indicates the number of neurons. Tiling percentage indicates the fraction of samples in which the Wave axon innervates the corresponding neuromere. (K, L) Quantification of axon extension toward the posterior end in A2 (K) and A6 (L) Wave neuron measured in MultiColor FlpOut (MCFO) images, following knockdown (KD) and overexpression of DFz2, respectively. **: p<0.01, ***: p<0.001, Welch’s t-test with Bonferroni correction. (M) Summary of DFz2 KD and overexpression phenotypes.
Figure 1—figure supplement 1
R60G09-GAL4 consistently targets Wave neurons from embryonic to larval stages.
(A) R60G09-GAL4 targets Wave neurons. Double labeling of MB120B-spGAL, a previously characterized Wave-specific GAL4, and newly identified R60G09-GAL4 and R77H11-LexA in 3rd instar larvae shows that these three lines commonly target Wave neurons (arrows). Scale bar = 50 μm. (B–D) R60G09-GAL4 consistently labels Wave neurons from embryonic to larval stages. (B) Expression driven by R60G09-GAL4 in 12 hr AEL (hAEL) (Stage 15) embryos. At this stage, the expression is seen in Wave neurons (white arrows) and putative dMP2 neurons (black arrows), which undergo apoptosis at later stages (except in segments A6-A8, Miguel-Aliaga and Thor, 2004). Stacked image. Scale bar = 50 μm. (C) Expression in 16 hAEL (Stage 16/17) embryos. White arrows: Wave neurons, black arrows: putative dMP2 neurons, gray arrows: degenerating corpses of putative dMP2 neurons. Stacked image. Scale bar = 50 μm. (D) Expression in 20 hAEL (Stage 17) embryos. White arrows indicate Wave neurons. Note that dMP2 neurons (except in segments A6-A8) are no longer present, and the GAL4-driven expression is now largely confined to Wave neurons at this stage. Stacked image. Scale bar = 50 μm. (E) Expression in 3rd instar larvae. In the ventral nerve cord (VNC), the GAL4-driven expression is confined to Wave neurons (white arrows) and a pair of ascending neurons in the T2 segment. Note that the GAL4-driven expression in dMP2 neurons (including A6-A8) is absent in larval stages. Stacked image. Scale bar = 50 μm.
Figure 1—figure supplement 2
Identification of candidate Wnt receptors and ligands that regulate anterior-posterior (A-P) Wave axon guidance.
(A–D) Identification of candidate receptors. R60G09-GAL4 was used to drive expression of UAS-RNAi constructs for Wnt receptors and CD4-GCaMP6f in Wave neurons. Since the expression of CD4-GCaMP6f visualizes all Wave axons which fasciculate together to form an axon bundle, the morphology of individual Wave axons cannot be assessed in general by this method. However, since it is known from previous single-cell analyses that the most posterior part of the Wave axon bundle contains only the axon of A6-Wave (Takagi et al., 2017), we focused on this part of the axon bundle (white arrows) to study how manipulation of Wnt receptor expression affects its morphology. As compared to the control (A), DFz2 KD [TRiP] (B) results in posterior elongation while DFz4 KD [TRiP] (C) results in shortening of this axon branch. Arrowhead: cell body of A6-Wave. Dotted boxes indicate the abnormal extension/shortening of the axon. Scale bar = 50 μm. (D) Quantification of the ending points of A6-Wave (p-Wave) axon extension in R60G09-GAL4>UAS-RNAi [TRiP] animals. The numbers shown in the bar charts represent the number of neurons (i.e. Wave neurons from left or right hemisphere). p-Values were calculated by Chi-square test (p=2.38 × 10–6) followed by Haberman’s adjusted residual analysis (α=0.0019; see Materials and methods details). Statistically significant groups are annotated with asterisks. (E) Identification of candidate ligands. Quantification of the ending points of A5-Wave (p-Wave) axon extension in each mutant line. MB120B-spGAL4 was used for DWnt5400 mutant and R77H11-GAL4 for all the other mutant lines to express CD4-GCaMP6f. p-Values were calculated by Chi-square test (p=0.024) followed by Haberman’s adjusted residual analysis (α=0.0064). Other indications are the same as in D. (F, F’) Ectopic posterior extension of A2-Wave axon by DFz2 KD with an independent RNAi line. (F) Lateral view of a single Wave clone in which DFz2 was knocked down with the KK RNAi line, visualized by mosaic analyses in 3rd instar larvae. As was observed with the TRiP RNAi line (see Figure 1B and D), the axon was ectopically extended toward the posterior (dotted box). The green channel shows the Wave neuron, and the blue channel shows anti-HRP staining that visualizes the neuropil. White arrows indicate axon processes. Scale bar = 50 μm. (F’) Quantification of the ectopic extension. Normalized posterior axon lengths were quantified in A2-Wave and categorized into normal (length <1) and abnormally extended (length >1) neurons. p-Value was calculated by one-tailed Fisher’s exact test (p=0.0455). Other indications are the same as in D. (G, G’) Posterior extension of A6-Wave is shortened by DFz4 KD with two RNAi lines (TRiP and KK). (G) A6-Wave (p-Wave) axon extension in control (left) and DFz4 KD (right, KK) animals. Note that the posterior extension of A6-Wave axons can be specifically examined in R60G09-GAL4>CD4-tdGFP animals, since only A6 Wave neurons extend axons beyond A6. Arrowhead: cell body of A6-Wave. Scale bar = 50 μm. (G’) Quantification of the ending points of A6-Wave (p-Wave) axon extension in control, DFz4 KD (TRiP), and DFz4 KD (KK) animals. p-Values were calculated by Chi-square test (p=0.0053) followed by Haberman’s adjusted residual analysis (α=0.0085). Other indications are the same as in D.
Figure 1—figure supplement 3
Effect of DFz2 knockdown (KD) on Wave dendrite extension.
Tiling profile of A2-Wave to A6-Wave dendrites in control and DFz2 knocked-down [TRiP #67863] animals. n indicates the number of neurons. Tiling percentage indicates the fraction of samples (single Wave neurons) whose Wave dendrite innervated the corresponding neuromere.
Figure 2
Ectopic synapses are formed in extended Wave axon projection.
(A) Dorsal view of a ventral nerve cord containing a single labeled A4-Wave (green) and stained with a presynaptic marker Bruchpilot (Brp) (magenta). Scale bar = 50 µm. (B) Enlarged images of neuromere A5. White and black arrowheads indicate Brp+ and Brp- boutons, respectively. Scale bar = 5 µm. (C) Number of A4-Wave synapses (Brp+ boutons) in each neuromere. (D) Number of all A4-Wave boutons in each neuromere. (E) Fraction of A4-Wave synapses over all boutons in each neuromere.
Figure 3
DFz4 specifically promotes axon extension toward the posterior end in A6-Wave.
(A, B) DFz4 knockdown (KD) A2 (A) and A6 (B) Wave neuron using the TRiP RNAi line. Note the shortening of the A6 axon toward the posterior end (dotted box in B, see Figure 1B and C for control). Scale bar = 50 µm. (C–G) Tiling profile of A2 to A6 Wave axons in control and DFz4 knocked-down animals. n indicates the number of neurons. Tiling percentage indicates the fraction of samples (single Wave neurons) whose Wave axon innervated the corresponding neuromere. The same analyses as in Figure 1F–J but for DFz4 knocked-down animals. The control data in Figure 1F–J are shown as references. (H, I) Quantification of axon extension toward the posterior end in A2 (H) and A6 Wave (I) measured in MultiColor FlpOut (MCFO) images, following KD and overexpression of DFz4, respectively. *: p<0.05, Welch’s t-test with Bonferroni correction. The control data shown in Figure 1K and L are reused for this analysis. (J) Summary of DFz4 KD and overexpression phenotypes.
Figure 4
DWnt4 regulates anterior-posterior (A-P) extension of Wave axons.
(A–C) DWnt4 promotes posterior axon extension of p-Wave. (A, B) Examples of axon morphologies toward the posterior end (white arrows) in control (A) and DWnt4C1/EMS23 mutant (B) animals. The posterior end of the axon, derived from p-Wave, is shortened in DWnt4 mutant. Dotted boxes indicate the abnormal shortening of the axon. (C) Quantification of A6-Wave axon extension to segment A6 or A7. n=6 (control) and 3 (DWnt4C1/EMS23) neurons, respectively: p=0.0119, Fisher’s exact test. (D–G) DWnt4 regulates axon extension of a-Wave. (D) A2-Wave in DWnt4C1/EMS23 is visualized using heat-shock FlpOut. Note the elongation of its axon toward the posterior end, as well as the shortening of its axon toward the anterior end (dotted boxes). Scale bar = 50 µm. See Figure 1B for control. (E) Tiling profile of A2-Wave axons in control and DWnt4C1/EMS23 mutant animals. n indicates the number of neurons. Tiling percentage indicates the fraction of samples (single Wave neurons) whose Wave axon innervated the corresponding neuromere. The control data in Figure 1F are reused and shown as references for different comparisons. (F, G) Quantification of the relative length (see Materials and methods details) of the anterior and posterior fraction of A2-Wave axons between control (visualized by MultiColor FlpOut [MCFO]) and mutant (visualized by heat-shock FlpOut) animals. (F) Quantification of the anterior fraction of A2-Wave axons. (G) Quantification of the posterior fraction of A2-Wave axons. n indicates the number of neurons. *: p<0.05, **: p<0.01, Welch’s t-test. The control data are replotted from Figure 1K for different comparisons. (H) Summary of DWnt4 phenotypes.
Figure 5 with 3 supplements
Complementary graded expression of DWnt4 and DFz2 along the anterior-posterior (A-P) axis.
(A, B) DWnt4-GFP cells in the embryonic CNS. (A) Distribution of DWnt4-GFP cells. Nuclear-localized GFP is expressed under the regulation of Wnt4MI03717-Trojan-GAL4. anti-Elav counterstaining was performed to label all neurons and normalize the GFP signal. GFP expression is stronger in posterior than anterior ventral nerve cord (VNC). Scale bar = 50 µm. (B) Quantification of normalized GFP signals with respect to Elav signals in each neuromere (where 1 denotes the maximum). n=5 animals. Not having a common alphabet indicated above the plots between groups indicates statistical significance (α=0.05, Tukey’s HSD test). (C, D) Expression of DWnt4 protein in the neuropil of each neuromere. (C) DWnt4 and HRP immunostaining. Scale bar = 10 μm. (D) Normalized DWnt4/HRP signals in each neuromere are calculated. n=18 samples from 9 animals *: p<0.05, ***: p<0.001, paired t-test (comparison to A8). (E, F) Expression of DFz2 protein in the neuropil of each neuromere. (E) DFz2 and HRP immunostaining. Scale bar = 10 μm. (F) Normalized DFz2/HRP signal in each neuromere. n=16 samples from 8 animals *: p<0.05, **: p<0.01, paired t-test (comparison to A2). (G) Model of segment-specific axon guidance in Wave neurons. DWnt4 serves as a graded axon guidance cue (concentrated toward the posterior end of VNC) and is recognized by DFz2/DFz4 receptors as repulsive/attractive cue, respectively. DFz2 functions in both a-Wave and p-Wave, whereas DFz4 functions selectively in p-Wave.
Figure 5—figure supplement 1
DWnt4-GFP gradient is retained in 3rd instar larvae.
(A, A') DWnt4-GFP cells in the larval CNS. Nuclear-localized GFP is expressed under the regulation of Wnt4MI03717-Trojan-GAL4. (A) GFP-positive cells are more abundant in posterior than anterior ventral nerve cord (VNC). Scale bar = 100 µm. (A') GFP-positive cells are Elav-positive, indicating that these cells are neurons. Scale bar = 10 µm. (B) Expression of DWnt4 in the CNS shows an anterior-posterior gradient. Normalized GFP signals with respect to Elav signals in each neuromere are calculated. n=5 animals.
Figure 5—figure supplement 2
Expression of DWnt4 and DFz2 is suppressed in mutants.
(A, B) Expression of DWnt4 protein in the neuropil of each neuromere. (A) The fluorescence of anti-DWnt4 antibody in control (yw) and mutant (DWnt4EMS23) embryos is shown (with the same contrast for each channel in each group). Scale bar = 10 μm. (B) Comparison of DWnt4 signal between control (yw) and mutant (DWnt4EMS23) embryos in each neuromere. ***: p<0.001, Welch’s t-test. (C, D) Expression of DFz2 protein in the neuropil of each neuromere. (C) The fluorescence of anti-DFz2 antibody in control (yw) and DFz2 KD (elav-GAL4>UAS-DFz2-RNAi [KK]) embryos is shown (with the same contrast for each channel in each group). Scale bar = 10 μm. (D) Comparison of DFz2 signal between control (yw) and DFz2 KD (elav-GAL4>UAS-DFz2-RNAi [KK]) embryos in each neuromere. *: p<0.05, **: p<0.01, ***: p<0.001, Welch’s t-test.
Figure 5—figure supplement 3
DFz2 gradient is bidirectional.
Normalized DFz2/HRP signal in each neuromere (same as in Figure 5F) but with statistical comparison to A8/9. n=16 samples from 8 animals *: p<0.05, **: p<0.01, paired t-test.
Figure 6
DFz2 knockdown (KD) in Wave neurons alters motor commands in vivo.
(A) Schematic of optogenetics assay in vivo. (B–B'') Comparison of the larval behavior between LED OFF and ON conditions in control animals. LED illumination decreases stride duration of forward locomotion (i.e. induces fast-forward locomotion) (B), induces backward locomotion (B'), and induces rolling (B''). n=52 animals. ***: p<0.001, Wilcoxon’s signed-rank test. (C–C'') Comparison of the larval behavior between LED OFF and ON conditions in DFz2 knocked-down [TRiP] animals. LED illumination decreases stride duration of forward locomotion (i.e. induces fast-forward locomotion) (C), does not induce backward locomotion (C'), and induces rolling (C''). n=18 animals. **: p<0.01, ***: p<0.001, Wilcoxon’s signed-rank test. (D, E) Comparison of the stride duration of forward locomotion between control and DFz2 knocked-down animals in LED OFF (D) and ON (E) conditions. The stride duration showed no significant difference in LED OFF period (D) but was shorter in DFz2 knocked-down animals in LED ON period (E). Mean ± SD stride durations: 0.85±0.32 (Ctrl, OFF), 0.71±0.22 (Ctrl, ON), 0.71±0.21 (DFz2 KD, OFF), 0.50±0.12 (DFz2 KD, ON). ***: p<0.001, Mann-Whitney’s U-test. Note that the stride durations of the control and DFz2 KD animals are slightly different in the OFF condition, although this is not statistically significant. In addition, the effect size of Wave activation on mean stride duration is –0.14 (s) in control while –0.21 (s) in DFz2 KD, which we interpret as DFz2 KD resulting in stronger fast-forward locomotion upon Wave activation.
Figure 7
Modulation of tactile-evoked behavior by DFz2 knockdown (KD) in Wave neurons.
(A) Left panel: Scheme of gentle-touch assay using von Frey filament. Right panel: Stereotypic behavioral responses induced by the gentle head touch, categorized according to the Kernan score (Kernan et al., 1994). (B–C”) Alteration in behavior responses upon DFz2 KD using UAS-DFz2-RNAi [KK] (B–B’’) or UAS-DFz2-RNAi [GD] (C–C’’). (B, C) Distribution of the Kernan score. Driver control; R60G09-GAL4/+, effector control; UAS-DFz2-RNAi/+, and experimental group (R60G09-GAL4>UAS-DFz2-RNAi). n=40 trials each. (B’, C’) Fraction of behavioral responses classified as backward (scores 3 and 4) and turning (score 2). The numbers indicate the trials that induced the classified response. *: p<0.05, **: p<0.01, ***: p<0.001, Fisher’s exact test with Bonferroni correction. (B’’, C’’) Quantifications of stride duration of forward locomotion following gentle touch. Note that smaller stride duration indicates faster forward locomotion. n=36 (driver control), 40 (effector control), 39 (B’’), and 40 (C’’) (experimental) trials. *: p<0.05, ***: p<0.001, Steel-Dwass test. (D) Summary of the current study.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | yw | Bloomington Drosophila Stock Center | RRID:BDSC_6598 | |
| Strain, strain background (D. melanogaster) | DWnt4C1 | Bloomington Drosophila Stock Center; Cohen et al., 2002 | RRID:BDSC_6651 | Null allele of DWnt4 |
| Strain, strain background (D. melanogaster) | DWnt4EMS23 | KYOTO Drosophila Stock Center; Cohen et al., 2002 | KYOTO:108–974 | Null allele of DWnt4 |
| Strain, strain background (D. melanogaster) | DWnt5400 | Bloomington Drosophila Stock Center; Fradkin et al., 2004 | RRID:BDSC_64300 | Null allele of DWnt5 |
| Strain, strain background (D. melanogaster) | DWnt6KO | Bloomington Drosophila Stock Center; Doumpas et al., 2013 | RRID:BDSC_76311 | Deletion of exon 1 of DWnt6 |
| Strain, strain background (D. melanogaster) | DWnt8KO1 | Bloomington Drosophila Stock Center; Gordon et al., 2005 | RRID:BDSC_38407 | Knockout allele of DWnt8 (aka wntD) |
| Strain, strain background (D. melanogaster) | UAS-CD4-tdGFP | Bloomington Drosophila Stock Center | RRID:BDSC_35836 | A membrane-fused GFP |
| Strain, strain background (D. melanogaster) | UAS-CD4::GCaMP6f | Takagi et al., 2017 | A membrane-fused GCaMP | |
| Strain, strain background (D. melanogaster) | MCFO-6 | Bloomington Drosophila Stock Center; Nern et al., 2015 | RRID:BDSC_64090 | MCFO-6 |
| Strain, strain background (D. melanogaster) | 20XUAS >dsFRT > -CsChrimson::mVenus, pBPhsFlp2::Pest | Takagi et al., 2017 | FLP-Out optogenetics construct | |
| Strain, strain background (D. melanogaster) | MB120B-spGAL4 | Takagi et al., 2017 | GAL4.AD and GAL4.DBD combination specific to Wave neurons | |
| Strain, strain background (D. melanogaster) | R60G09-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_46441 | GAL4 line targeting Wave neurons |
| Strain, strain background (D. melanogaster) | R77H11-GAL4 | Bloomington Drosophila Stock Center; Masson et al., 2020 | RRID:BDSC_39983 | Targets Wave neurons |
| Strain, strain background (D. melanogaster) | R77H11-LexA | Bloomington Drosophila Stock Center | RRID:BDSC_54720 | LexA version of R77H11 enhancer |
| Strain, strain background (D. melanogaster) | Wnt4MI03717-Trojan-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_67449 | GAL4 insertion near Wnt4 locus |
| Strain, strain background (D. melanogaster) | elav-GAL4[3E1] | Davis et al., 1997 | Targets all neurons | |
| Strain, strain background (D. melanogaster) | UAS-mCherry.VALIUM10 | Bloomington Drosophila Stock Center | RRID:BDSC_35787 | Control stock for RNAi reporter lines |
| Strain, strain background (D. melanogaster) | UAS-DFz-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_34321 | RNAi to knock down DFz |
| Strain, strain background (D. melanogaster) | UAS-DFz2-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_67863 | RNAi to knock down DFz2 |
| Strain, strain background (D. melanogaster) | UAS-DFz3-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_44468 | RNAi to knock down DFz3 |
| Strain, strain background (D. melanogaster) | UAS-DFz4-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_64990 | RNAi to knock down DFz4 |
| Strain, strain background (D. melanogaster) | UAS-drl-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_39002 | RNAi to knock down Drl |
| Strain, strain background (D. melanogaster) | UAS-Drl-2-RNAi [TRiP] | Bloomington Drosophila Stock Center | RRID:BDSC_55893 | RNAi to knock down Drl-2 |
| Strain, strain background (D. melanogaster) | UAS-Drl-2-RNAi [KK] | Vienna Drosophila Resource Center | VDRC:102192 | RNAi to knock down Drl-2 |
| Strain, strain background (D. melanogaster) | UAS-smo-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_43134 | RNAi to knock down Smo |
| Strain, strain background (D. melanogaster) | UAS-Corin-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_41721 | RNAi to knock down Corin |
| Strain, strain background (D. melanogaster) | UAS-DFz2-RNAi [GD] | Vienna Drosophila Resource Center | VDRC:44390 | RNAi to knock down DFz2 |
| Strain, strain background (D. melanogaster) | UAS-DFz2-RNAi [KK] | Vienna Drosophila Resource Center | VDRC:108998 | RNAi to knock down DFz2 |
| Strain, strain background (D. melanogaster) | UAS-DFz4-RNAi [KK] | Vienna Drosophila Resource Center | VDRC:102339 | RNAi to knock down DFz4 |
| Strain, strain background (D. melanogaster) | UAS-DFz2-ORF | FlyORF | FlyORF:F001187 | ORF line for DFz2 overexpression |
| Strain, strain background (D. melanogaster) | UAS-DFz4-ORF | FlyORF | FlyORF:F001662 | ORF line for DFz4 overexpression |
| Strain, strain background (D. melanogaster) | LexAop-RCaMP2 | KYOTO Drosophila Stock Center | KYOTO:118796 | Takagi et al., 2017 |
| Antibody | Rabbit polyclonal anti-GFP | Frontier Institute | Af2020; RRID:AB_10615238 | 1/1000 |
| Antibody | Mouse monoclonal anti-Fas2 (1D4) | Developmental Studies Hybridoma Bank | RRID:AB_528235 | 1/10 |
| Antibody | Rat monoclonal anti-Elav (7E8A10) | Developmental Studies Hybridoma Bank | RRID:AB_528218 | 1/10 |
| Antibody | Mouse monoclonal anti-Repo (8D12) | Developmental Studies Hybridoma Bank | RRID:AB_528448 | 1/5 |
| Antibody | Mouse monoclonal anti-Brp (nc82) | Developmental Studies Hybridoma Bank | RRID:AB_2314866 | 1/100 |
| Antibody | Guinea pig polyclonal anti-GFP | Frontier Institute | Af1180; RRID:AB_2571575 | 1/1000 |
| Antibody | Rabbit monoclonal anti-HA (C29F4) | Cell Signaling Technology | RRID:AB_1549585 | 1/1000 |
| Antibody | Mouse monoclonal anti-V5 | Invitrogen | R960-25; RRID:AB_2556564 | 1/500 |
| Antibody | Rat monoclonal anti-FLAG | Novus Biologicals | NBP1-06712; RRID:1625981 | 1/500 |
| Antibody | Rabbit polyclonal anti-DWnt4 | Cohen et al., 2002; Gift from M Sato | 1/100 | |
| Antibody | Rabbit anti-DFz2 | Packard et al., 2002; Gift from G Alegre, V Budnik, and T Thomson | 1/100 | |
| Antibody | Goat polyclonal anti-rabbit IgG Alexa Fluor 488 | Invitrogen | A-11034; RRID:AB_2576217 | 1/300 |
| Antibody | Goat polyclonal anti-rabbit IgG Cy3 | Invitrogen | A-10520; RRID:AB_10563288 | 1/300 |
| Antibody | Goat polyclonal anti-mouse IgG Alexa Fluor 555 | Invitrogen | A-21424; RRID:AB_141780 | 1/300 |
| Antibody | Goat polyclonal anti-mouse IgG Cy5 | Invitrogen | A-10524; RRID:AB_2534033 | 1/300 |
| Antibody | Goat polyclonal anti-rat IgG Alexa Fluor 488 | Invitrogen | A-11006; RRID:AB_141373 | 1/300 |
| Antibody | Goat polyclonal anti-HRP Cy5 | Jackson ImmunoResearch | 123-175-021 | 1/300 |
| Antibody | Goat polyclonal anti-HRP Alexa Fluor 647 | Jackson ImmunoResearch | 123-605-021; RRID:AB_2338967 | 1/300 |
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Segment-specific axon guidance by Wnt/Fz signaling diversifies motor commands in Drosophila larvae
eLife 13:RP98624.
https://doi.org/10.7554/eLife.98624.3
