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1 Supplementary Fig. 1. Dendrite length is not secondary to body length. Dendrite growth proceeds independently of the rate of body growth and decreases in rate in adults. n 20 on dendrite measurement, n 12 on body measurement. Error bars: standard error of the mean (s.e.m).
2 Supplementary Fig. 2. Dendrite length is not dependent on axon guidance. Dendrite length is not significantly different in unc-6 mutant animals displaying axon guidance phenotypes from unc-6 mutant animals expressing a wild-type axon trajectory. n 35. Error bars: standard error of the mean (s.e.m).
3 Supplementary Fig. 3. UNC-40 localizes to the dendrite in an unc-6-dependent manner. (a, b) Lateral views of L3 animals expression Ex[Pmig-13::unc-40::gfp]. (a) UNC-40::GFP expression in wild-type animals shows preferential localization to the dendrite. (b) UNC-40::GFP expression in unc-6 mutants suppresses preferential localization to the dendrite. (c) Intensity ratio of dendrite to axon UNC-40::GFP fluorescence in wild-type, unc-6 mutant and unc-6 mutant expressing unc-6 in dorsal muscle (Ex[Punc-129::unc-6]) compared to muis42 (Pmig-13::mig-13::gfp). All animals are L3 stage, all pictures are lateral view with ventral down and anterior to the left, all quantification from anterior edge of soma to anterior tip of dendrite. Scalebar 20 μm. n 20. ***p<0.001, Mann-Whitney U test.
4 Supplementary Fig. 4. Axon guidance phenotypes in unc-6 overexpression experiments. Compared to the penetrance of axon guidance phenotypes in unc-6 mutant animals, the expression of a membrane-tethered unc-6 construct (Ex[Punc-6::unc-6:: nlg-1tm::mcherry]) had no effect, while the expression of unc-6 in dorsal body wall muscles (Ex[Punc-129::unc-6]) doubled the penetrance of dorsal axon misguidance. n 73. Error bars: standard error of the mean (s.e.m). ***p<0.001, C 2 -test.
5 Supplementary Fig. 5. par-4 acts in the same pathway as unc-6 and unc-40. Graphs of dendrite lengths in wild-type, unc-40, par-4, unc-40; par-4, unc-6 and par-4; unc-6 mutants at L4 (a) and adult stages (b). n 30. Error bars: standard error of the mean (s.e.m.). ***p<0.001, student s t-test.
6 Supplementary Fig. 6. unc-40 regulates par-4 via the intracellular P2 motif. Quantification of gain of function phenotypes in AVM in adult worms in myr::unc-40, myr::unc-40δp1, myr::unc-40δp2 and myr::unc- 40ΔP3 animals and their suppression in unc-34, unc-115 and par-4 mutants. n 90. Error bars (not visible): standard error of proportion. *p<0.05, ***p<0.001, C 2 -test.
7 Supplementary Fig. 7. UNC-6 plays multiple roles in the development of one neuron. (a) During embryonic development, UNC-5 repels the DA9 axon from the graded ventral UNC-6 source; during larval development and adulthood, UNC-6 regulates dendrite outgrowth and exclusion of presynaptic cargo from the dendrite via the UNC-40 and UNC-5 receptors respectively. (b) Dendrite outgrowth is mediated by short-range UNC-6 signal acting via a novel pathway including UNC-40 and PAR-4.
8 Supplementary Table 1. In all tables below, n represents the number of animals quantified in each category. Mean represents mean length of dendrite in that category in μm, s.e.m. is the standard error of the mean. Where p is indicated it represents the p-value outcome of a student s t-test or Pearson s C 2 -test as specified. Unless indicated otherwise, the p-value in a category is in a comparison of that population to wild-type. If the comparison is with a different genotype, the genotype is named before the p-value. In tables in which such comparisons occur, wild-type (wt) is stated as well. Fig. 1g wt L1 L2 L3 L4 YA A n mean s.e.m Fig. 2e wt L1 L2 L3 L4 n mean s.e.m unc-6 L1 L2 L3 L4 n mean s.e.m unc-5 L1 L2 L3 L4 n mean s.e.m unc-40 L1 L2 L3 L4 n mean s.e.m unc-34 L1 L2 L3 L4 n mean s.e.m unc-115 L1 L2 L3 L4 n mean s.e.m Fig. 2f. wt unc-6 unc-5 unc-40 unc-34 unc-115 n mean s.e.m p 1.34x x x x10-4 Fig. 3a wt Pmig-13::unc-40 unc-40 unc-40;pmig-13::unc-40 unc-6 unc-6;pmig-13::unc-40 n mean s.e.m p x x x10-5 Fig. 4a wt unc-6 unc-6;punc-6::unc-6::nlg-1tm::mcherry unc-6; Punc-129::unc-6 Punc-6::unc-6::nlg-1TM::mCherry n mean s.e.m p 3.06x x10-5 Fig. 5c wt par-4 par-4;pitr-1::par-4::gfp Pitr-1::par-4::gfp n mean s.e.m p 3.47x x10-12
9 Fig. 6a. wt par-4 unc-6 par-4; unc-6 unc-40 unc-40; par-4 unc-34 unc-34, par-4 unc-115 par-4; unc-115 n mean s.e.m p wt wt unc-6 wt unc-40 wt unc-34 wt par x x x x x x ; unc Fig. 6b. wt unc-6 unc-40 par-4 unc-6;pitr-1::par-4::gfp unc-40;pitr-1::par-4::gfp par-4;pmig-13::unc-40 n mean s.e.m p wt wt wt unc-6 wt wt 1.34x x x x x x10-4 wt unc-40 par x x Fig. 6c. wt unc-115 par-4 par-4; unc-115 Pitr-1::par-4::gfp unc-115; Pitr-1::par-4::gfp. n mean s.e.m p wt wt par-4 wt Pitr-1::par-4::gfp 2.00x x ; 1.68x x10-13 unc-115 unc Fig.7c. wt par-4 unc-34 unc-34, par-4 n guidance 0/105 0/101 3/101 13/125 s.e.p p (χ2) unc Fig.7d. wt par-4 unc-34 unc-34, par-4 n guidance 0/105 0/101 0/101 8/125 s.e.p p (χ2) unc Supplementary Fig. 1. L1 L2 L3 L4 YA A n body length Mean body length s.e.m. body length Mean dendrite/ body length (Fig. 1g) Propagated error dendrite/body length Supplementary Fig. 2. Unc-6 dorsal axon Unc-6 axon misguided n mean s.e.m p unc-6 dorsal axon 0.75 Supplementary Fig. 3. Pmig-13::unc-40::gfp unc-6, Pmig-13::unc-40::gfp unc-6, Pmig-13::unc-40::gfp, Punc-129::unc-6 muis42 n median IQR p (Mann-Whitney U test) Pmig-13::unc-40::gfp unc-6, Pmig-13::unc-40::gfp unc-6, Pmig-13::unc-40::gfp
10 Supplementary Fig. 4.- see Supplementary Figure 4. Supplementary Fig. 5a. wt unc-6 par-4 par-4; unc-6 unc-40 unc-40; par-4 n mean s.e.m p 5.6x x x x x10-11 unc-6 unc Supplementary Fig. 5b. wt unc-6 par-4 par-4; unc-6 unc-40 unc-40; par-4 n mean s.e.m p 4.5 x x x x x10-20 par-4 unc Supplementary Fig. 6. genotype n gain of function s.e.p p ((χ2) myr::unc unc-34; myr::unc myr::unc-40 <.0001 unc-115; myr::unc myr::unc-40 <.0001 par-4; myr::unc myr::unc-40 <.0001 myr::unc-40δp myr::unc unc-34;myr::unc-40δp myr::unc-40δp unc-115; myr::unc-40δp myr::unc-40δp1 <.0001 par-4; myr::unc-40δp myr::unc-40δp1 <.0001 unc-115;myr::unc-40δp myr::unc-40δp myr::unc unc-34; myr::unc-40δp myr::unc-40δp2 <.0001 unc-115; myr::unc-40δp myr::unc-40δp par-4; myr::unc-40δp myr::unc-40δp2 1.0 unc-115; myr::unc-40δp myr::unc-40δp myr::unc
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